SIMPLE TAPE MODIFIED OLIGONUCLEOTIDE AIMED AT A NUCLEIC ACID THAT ENCODES HUNTINGTIN AND CAPABLES TO

专利摘要:
compound, composition comprising said compound and uses thereof. provided herein are methods, compounds and compositions for reducing mRNA huntingtin and protein expression in an animal. such methods, compounds and compositions are useful for treating, preventing, delaying, or ameliorating huntington's disease, or a symptom thereof.
公开号:BR112012005448B1
申请号:R112012005448-1
申请日:2010-09-10
公开日:2021-09-14
发明作者:Susan M. Freier；Gene Hung；C. Frank Bennett；Holly Kordasiewicz；Lisa Stanek；Don W. Cleveland；Lamya Shihabuddin；Seng H. Cheng
申请人:Ionis Pharmaceuticals, Inc；
IPC主号:

专利说明:

Sequence Listing
[0001] The present application is being filed together with a String Listing in electronic format. The sequence listing is provided as a file titled BIOL0113WOSEQ.txt created on September 8, 2010, which is 456Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. field of invention
[0002] Methods, compounds and compositions for reducing huntingtin mRNA and protein expression in an animal are provided. Such methods, compounds and compositions are useful, for example, to treat, prevent or ameliorate Huntington's disease. Fundamentals
[0003] Huntington's disease (HD) is a devastating autosomal dominant neurodegenerative disease caused by the expansion of the CAG trinucleotide encoding an abnormally long polyglutamine (PolyQ) tract in the huntingtin protein. The Huntington's disease gene was first mapped in 1993 (The Huntington's Disease Collaborative Research Group. Cell. 1993, 72:971-83), which consists of a gene, IT15, which contained a polymorphic trinucleotide repeat that is expanded and unstable on HD chromosomes. Although CAG repeats in the normal size range, it is usually inherited as Mendelian alleles, expanded HD repeats are unstable through meiotic transmission and are observed to be expanded beyond the normal size range (6 to 34 repeat units) in patients with HD.
[0004] Both normal and variant huntingtin protein are located primarily in the cytoplasm of neurons (DiFiglia et al., Neuron 1995, 14:1075-81). As a result of excessive polyglutamine length, huntingtin protein forms aggregates in the cytoplasm and nucleus of CNS neurons (Davies et al., Cell 1997, 90:537-548). Both transgenic animals and genetically engineered cell lines were used to investigate the effects of expanded polyQ repeats on huntingtin localization and processing. However, it is still uncertain whether aggregate formation per se is the essential cytotoxic step or a consequence of cell dysfunction.
[0005] HD is characterized by progressive chorea, psychiatric changes, and intellectual decline. This dominant disorder affects men and women equally and occurs in all races (Gusella and MacDonald, Curr. Opin. Neurobiol. 1995 5:656-62). Symptoms of HD occur due to the death of neurons in many brain regions, but it is most evident in the striatum, particularly in the caudate nucleus, which undergoes a progressive gradient of cell loss that ultimately decimates the entire structure. Although the huntingtin-encoding gene is ubiquitously expressed (Strong, TV et al., Nat. Genet. 1995, 5:259-263), selective cell loss and fibrillary astrocytosis are observed in the brain, particularly in the caudate and striatal putamen and in the cerebral cortex of patients with HD (Vonsattel, JP. et al., Neuropathol. Exp. Neurol. 1985, 44:559-577) and, to a lesser extent, in the hippocampus (Spargo, E. et al., J Neurol. Neurosurg. Psychiatry 1993, 56:487-491) and the subthalamus (Byers, RK' et al., Neurology 1973, 23:561-569).
Huntingtin is crucial for abnormal development and may be related to a cell survival gene (Nasir et al., Human Molecular Genetics, Vol 5, 1431-1435). Normal huntingtin function remains incompletely characterized, but based on protein-protein interactions it appears to be associated with the cytoskeleton and required for neurogenesis (Walling et al., J. Neurosci Res. 1998, 54:301-8). Huntingtin is specifically cleaved during apoptosis by a key cystepine protease, apopain, known to play a pivotal role in apoptotic cell death. The cleavage band is intensified by longer polyglutamine tracts, suggesting that inappropriate apoptosis subordinates to HD.
[0007] Antisense technology is emerging as an effective means to reduce the expression of specific gene products and may therefore prove to be uniquely useful in various therapeutic, diagnostic and research applications for modulating huntingtin expression. (See, U.S. Patent Publication Nos. 2008/0039418 and 2007/0299027)
[0008] Antisense compounds for modulating huntingtin expression are disclosed in the aforementioned published patent applications. However, there remains a need for such additional compounds. Invention Summary
[0009] Methods, compounds and compositions for modulating huntingtin expression and treating, preventing, delaying or ameliorating Huntington's disease and/or a symptom thereof are provided. Brief Description of Figures Fig. 1:
[00010] The PK/PD ratio of huntingtin mRNA expression in intrastriatal tissue with ISIS 387898 concentration in mouse brain. C57/BL6 mice were administered a single bolus of 50 μg of ISIS 387898 and huntingtin bm mRNA expression as the tissue antisense oligonucleotide concentration was measured. The EC50 of ISIS 387898 was also calculated. Fig. 2:
[00011] Comparison of huntingtin mRNA expression in striatal tissue and ISIS 387898 concentrations at various time points. C57/BL6 mice were administered a single 50 μg bolus of ISIS 387898 and huntingtin mRNA expression as well as tissue antisense oligonucleotide concentration were measured. The duration of action (as measured by htt mRNA expression) of ISIS 387898 (dashed line) was observed to be even longer after concentration of the oligonucleotide (solid line) in the tissue. Fig. 3:
[00012] The PK/PD ratio of huntingtin mRNA expression in anterior cortex tissue with ISIS 387898 concentration in mouse brain. BACHD mice were given an intracerebroventricular infusion of 75 μg of ISIS 387898 for 2 weeks and huntingtin mRNA expression as well as tissue antisense oligonucleotide concentration were measured. The EC50 of ISIS 387898 was also calculated. Fig. 4:
[00013] Comparison of huntingtin mRNA expression in anterior cortex tissue and ISIS 387898 concentrations at various time points. BACHD mice were given 75 μg intracerebroventricular infusion of ISIS 387898 for 2 weeks and huntingtin mRNA expression as well as tissue antisense oligonucleotide concentration were measured. The duration of action (as measured by htt mRNA expression) of ISIS 387898 (dashed line) was observed to be even longer after concentration of the oligonucleotide (solid line) in the tissue. Fig. 5:
[00014] Comparison of huntingtin mRNA expression in posterior cortex tissue and ISIS 388241 concentrations at various time points. BACHD mice were given an intracerebroventricular infusion of 50 μg of ISIS 388241 for 2 weeks and huntingtin mRNA expression as well as tissue antisense oligonucleotide concentration were measured. The duration of action (as measured by htt mRNA expression) of ISIS 388241 (dashed line) was observed to be even longer after concentration of the oligonucleotide (solid line) in the tissue. Fig. 6:
[00015] Comparison of huntingtin mRNA expression in posterior cortex tissue and ISIS 443139 concentrations at various time points. BACHD mice were administered intracerebroventricular infusion of 50 μg of ISIS 443139 for 2 weeks and huntingtin mRNA expression as well as tissue antisense oligonucleotide concentration were measured. The duration of action (as measured by htt mRNA expression) of ISIS 443139 (dashed line) was observed to be even longer after concentration of the oligonucleotide (solid line) in the tissue. Fig. 7.
[00016] The effect of antisense oligonucleotide treatment on the motor performance of BACHD mice using the Rotarod assay. BACHD mice were treated with 50 μg/day of ISIS 388241 ICV or PBS for two weeks. Control groups of non-transgenic litters were similarly treated with ISIS 388241 or PBS. The Rotarod test acceleration was then performed. The animals were placed on the Rotarod at a speed of 2 RPM; the Rotarod accelerated to 40 RPM for 5 minutes. Duration until drop was recorded. Baseline values at 6 months of age were taken prior to treatment and the time points given are the age of the mice in which the trial was conducted. Bars represent duration to fall in seconds by BACHD mice treated with ISIS 388241 (black) or BACHD mice treated with PBS (streaked) and by non-transgenic litters treated with PBS (white). Mice treated with ISIS 388241 showed increased duration of fall, hence improved motor performance on Rotarod compared to PBS control. Fig. 8.
[00017] The effect of antisense oligonucleotide treatment on brain weight of R6/2 mice. Six-month-old R6/2 mice were treated with 50 μg/day of ISIS 388817 or ISIS 141923 control oligonucleotide ICV or PBS for 4 weeks. Control groups of non-transgenic litters were similarly treated with ISIS 388817 or PBS. A control group of eight-week-old pre-symptomatic R6/2 mice were included in the study and received no treatment. Bars represent brain weights of untreated eight-week-old R6/2 mice; the R6/2 mice treated with ISIS 141923; PBS-treated R6/2 mice; R6/2 mice treated with ISIS 388817; non-transgenic litters treated with PBS and non-transgenic litters treated with ISIS 388817. There was an increase in brain weight of R6/2 mice treated with ISIS 388817 compared to the PBS control. Fig. 9
[00018] Behavioral characterization of YAC128 mice treated with antisense oligonucleotide using the Open Field assay. Five-month-old YAC128 mice were treated with 50 μg/day of ISIS 388241 ICV or ISIS 141923 control oligonucleotide or PBS for 14 days. A control group of non-transgenic FVB/NJ were included in the study giving no treatment. The mice were placed in an open field arena that uses photobeam breaks to measure horizontal and vertical movement in a 30-minute testing session. Data were analyzed using Activity Monitor software to examine complete ambulatory movement within the arena and movement within the center of the arena as a measure of anxiety. Bars represent time in seconds spent in the center of the field by FVB/NJ mice, YAC128 mice treated with PBS, and YAC128 mice treated with ISIS 388241. YAC128 mice treated with ISIS 388241 spent more time in the center and were therefore considered less prone to anxiety than PBS control. Fig. 10
[00019] Behavioral characterization of YAC128 mice treated with antisense oligonucleotide using the Elevated Plus Maze assay. Five-month-old YAC128 mice were treated with 50 μg/day of ISIS 388241 ICV or ISIS 141923 control oligonucleotide or with PBS for 14 days. A control group of non-transgenic FVB/NJ litters were included as an untreated control. The mice were placed in the center of a mechanism that consisted of two open arms and two closed arms, each measuring 65 x 6.25 cm and elevated 50 cm above the ground. The mice's location in the mechanism and amount of time spent in open arms was recorded in a 5-minute test session as a measure of anxiety. The bars represent the percentage of time spent in the open arms by FVB/NJ control, YAC128 treated with PBS, and YAC128 mice treated with ISIS 388241. YAC128 mice treated with ISIS 388241 spent more time in the open arms and were therefore considered less prone to anxiety than PBS control. Detailed Description of the Invention
[00020] It is to be understood that both the preceding general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention as claimed. Here, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless otherwise stated. Furthermore, the use of the term "including" as well as other forms such as "includes" and "included" is not limiting. Also, terms such as "element" or "component" encompass both elements and components that comprise a unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
[00021] The headings section used here is for organizational purposes only and should not be construed as limiting the subject material described. All documents or portions of documents cited in this application, including, but not limited to, patents, patent applications, articles, books, and treaties, are expressly incorporated herein by reference to the portions of the document discussed herein, as well as in their entirety. Definitions
[00022] Unless specific definitions are provided, the nomenclature used in connection with and the procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession numbers and associated sequence information through databases such as the National Center for Biotechnology Information (NCBI) and other data referred to throughout the disclosure are incorporated by reference to portions of the document discussed herein, as well as in their entirety.
[00023] Unless otherwise indicated, the following terms have the following meanings:
[00024] "2'-O-methoxyethyl" (also 2'-MOE and 2'-O(CH2)2-OCH3) refers to an O-methoxy-ethyl modification of the 2' position of a furosyl ring . A sugar modified by 2'-O-methoxyethyl is a modified sugar.
[00025] The "2'-O-methoxyethyl nucleotide" means a nucleotide comprising a sugar moiety modified by 2'-O-methoxyethyl.
[00026] "5-methylcytosine" means a cytosine modified by a methyl group attached to the 5' position. A 5-methylcytosine is a modified nucleobase.
[00027] “Active pharmaceutical agent” means the substance or substances in a pharmaceutical composition that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments a huntingtin-targeted antisense oligonucleotide is an active pharmaceutical agent.
[00028] "Active target region" or "target region" means a region to which one or more active antisense compounds are targeted. "Active antisense compounds" means antisense compounds that reduce nucleic acid levels or target protein levels.
[00029] “Administered concurrently” refers to the co-administration of two agents in any manner in which the pharmacological effects of both are manifested in the patient at the same time. Concomitant administration does not require both agents to be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not manifest themselves at the same time. The effects only need to overlap for a period of time and need not be coextensive.
[00030] “Administration” means providing a pharmaceutical agent to an individual and includes, but is not limited to, administration by a medical professional and self-administration.
[00031] “Improvement” refers to a decrease in at least one indicator, sign or symptoms of an associated disease, disorder or condition. The severity of indicators can be determined by subjective or objective measures, which are known to those skilled in the art.
[00032] "Animal" refers to a human or non-human animal, which includes, but is not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, which include, but are not limited to up to, monkeys and chimpanzees.
[00033] "Antisense activity" means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.
[00034] "Antisense compound" means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
[00035] "Antisense inhibition" means the reduction of target nucleic acid levels or target protein levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels or protein levels target in the absence of the antisense compound.
[00036] "Antisense oligonucleotide" means a single-stranded oligonucleotide having a nucleobase sequence that allows hybridization to a corresponding region or segment of a target nucleic acid.
[00037] "Bicyclic sugar" means a furosyl ring modified by bridging two non-geminal ring atoms. A bicyclic sugar is a modified sugar.
[00038] "Bicyclic nucleic acid" or "BNA" refers to a nucleoside or nucleotide in which the furanose portion of the nucleoside or nucleotide includes a bridge connecting two carbon atoms in the furanose ring, thereby forming a system of bicyclic ring.
[00039] "Coating structure" or "end cap portion" means chemical modifications, which have been incorporated into each end of an antisense compound.
[00040] "Chemically distinct region" refers to a region of an antisense compound that is in some way different from another region of the same antisense compound. For example, a region having 2'-O-methoxyethyl nucleotides is chemically different from a region having unmodified 2'-O-methoxyethyl nucleotides.
[00041] "Chimeric antisense compound" means an antisense compound that has at least two chemically distinct regions.
[00042] “Co-administration” means the administration of two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents can be in a single pharmaceutical composition or can be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents can be administered via the same or different route of administration. Co-administration encompasses parallel or sequential administration.
[00043] "Complementarity" means the ability to form pairs between nucleobases of a first nucleic acid and a second nucleic acid.
[00044] "Contiguous nucleobases" means nucleobases immediately adjacent to one another.
[00045] “Diluent” means an ingredient in a composition that loses pharmacological activity but is pharmaceutically necessary or desirable. For example, the diluent in an injected composition can be a liquid solution, eg, saline.
[00046] “Dose” means a specified amount of a pharmaceutical agent provided in a single administration or in a specified period of time. In certain embodiments, the dose can be administered in one, two or more boluses, tablets or injections. For example, in certain embodiments, when subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection, so two or more injections can be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses can be established as the amount of pharmaceutical agent per hour, day, week or month.
[00047] "Effective amount" means the amount of active pharmaceutical agent sufficient to effect a desired physiological result in an individual in need of the agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of individuals to be treated, the formulation of the composition, estimation of the individual's medical condition, and other relevant factors.
[00048] “Huntingtin nucleic acid” means any nucleic acid that encodes huntingtin. For example, in certain embodiments, a huntingtin nucleic acid includes a DNA sequence that encodes huntingtin, an RNA sequence transcribed from DNA that encodes huntingtin (including genomic DNA that comprises introns and exons), and an mRNA sequence that encodes huntingtin. “huntingtin mRNA” means an mRNA that encodes a huntingtin protein.
[00049] "Fully complementary" or "100% complementarity" means each nucleobase of a nucleobase sequence of a first nucleic acid having a complementary nucleobase in a second sequence of a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.
[00050] "Gapmer" means a chimeric antisense compound in which an inner region having a plurality of nucleosides that supports RNase H cleavage is positioned between the outer regions having one or more nucleosides, wherein the nucleosides comprising the region inner are chemically different from the nucleoside or nucleosides that comprise the outer regions. The inner region may be referred to as a "slack segment" and the outer regions may be referred to as "wing segments".
[00051] "Slack-Augmented" means a chimeric antisense compound having a slack segment of 12 or more contiguous 2'-deoxyribonucleosides positioned between and immediately adjacent to the 5' and 3' wing segments having one to six nucleosides.
[00052] "Hybridization" means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include an antisense compound and a target nucleic acid.
[00053] “Immediately adjacent” means that there are no intervening elements between the immediately adjacent elements.
[00054] “Individual” means a human or non-human animal selected for treatment or therapy.
[00055] "Internucleoside bond" refers to the chemical bond between nucleosides.
[00056] "Linked nucleosides" means adjacent nucleosides that are linked together.
[00057] "Disagreement" or "non-complementary nucleobase" refers to the case when a nucleobase of a first nucleic acid is not able to form pairs with the corresponding nucleobase of a second nucleic acid or target.
[00058] "Modified internucleoside linkage" refers to a substitution or any change of a naturally occurring internucleoside (i.e., a phosphodiester internucleoside linkage).
[00059] "Modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine or uracil. An "unmodified nucleobase" means the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
[00060] "Modified nucleotide" means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage or modified nucleobase. A "modified nucleoside" means a nucleoside having, independently, a modified sugar moiety or modified nucleobase.
[00061] "Modified oligonucleotide" means an oligonucleotide comprising at least one modified nucleotide.
[00062] “Modified sugar” refers to a substitution or change of a natural sugar.
[00063] "Motif" means a pattern of chemically distinct regions in an antisense compound.
[00064] "Natural-occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage.
[00065] "Natural sugar portion" means a sugar found in DNA (2'-H) or RNA (2'-OH).
[00066] "Nucleic acid" refers to molecules composed of monomeric nucleotides. A nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA). A nucleic acid can also comprise a combination of these elements into a single molecule.
[00067] "Nucleobase" means a heterocyclic portion capable of forming pairs with a base of another nucleic acid.
[00068] "Nucleobase sequence" means the order of contiguous nucleobases independent of any sugar, linkage or nucleobase modification.
[00069] "Nucleoside" means a nucleobase attached to a sugar.
[00070] "Nucleotide" means a nucleoside having a phosphate group covalently attached to the sugar portion of the nucleoside.
[00071] "Oligomeric compound" or "oligomer" means a polymer of linked monomeric subunits that are capable of hybridizing to at least one region of a nucleic acid molecule.
[00072] "Oligonucleotide" means a polymer of linked nucleosides each of which can be modified or unmodified, independent of each other.
[00073] "Parental administration" means administration by injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration or intracranial administration, for example, intrathecal or intracerebroventricular administration. Administration can be continuous or chronic or short or intermittent.
[00074] "Peptide" means a molecule formed by the linkage of at least two amino acids by amide bonds. Peptide refers to polypeptides and proteins.
[00075] “Pharmaceutical composition” means a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition can comprise one or more active pharmaceutical agents and a sterile aqueous solution.
[00076] "Pharmaceutically acceptable salts" means physiologically and pharmaceutically acceptable salts of antisense compounds, that is, salts that retain the desired biological activity of the oligonucleotide precursor and do not communicate unwanted toxicological effects thereto.
[00077] "Phosphorothioate bond" means a bond between nucleosides where the phosphodiester bond is modified by replacing one of the unbridged oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.
[00078] "Portion" means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a moiety is a defined number of contiguous nucleobases of an antisense compound.
[00079] “Avoiding” refers to delaying or preventing the onset or development of a disease, disorder or condition for a period of time from minutes to indefinitely. Avoidance also means reducing the risk of developing a disease, disorder or condition.
[00080] "Prodrug" means a therapeutic agent that is prepared in an inactive form that is converted to an active form within the body or cells of the body by the action of endogenous enzymes or other chemicals or conditions.
[00081] "Side effects" means other physiological responses attributable to a treatment than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased serum aminotransferase levels may indicate liver toxicity or abnormal liver function. For example, increased bilirubin may indicate toxicity or abnormality in liver function.
[00082] "Single-stranded oligonucleotide" means an oligonucleotide that is not hybridized to a complementary strand.
[00083] "Specifically hybridizable" refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while inhibiting minimal or no effects on nucleic acids non-target under conditions where specific binding is desired, that is, under physiological conditions in the case of in vivo assays and therapeutic treatments.
[00084] "Bleaching" or "targeted" means the process of designing and selecting an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.
[00085] "Target nucleic acid", "target RNA" and "target RNA transcript" all refer to a nucleic acid capable of being targeted by antisense compounds.
[00086] "Target segment" means the nucleotide sequence of a target nucleic acid to which an antisense compound is targeted. "5' target site" refers to the 5' most nucleotide of a target segment. "3' target site" refers to the 3' most nucleotide of a target segment.
[00087] "Therapeutically effective amount" means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.
[00088] "Treating" refers to the administration of a pharmaceutical composition to effect an alteration or amelioration of a disease, disorder or condition.
[00089] "Unmodified nucleotide" means a nucleotide composed of naturally occurring nucleobases, sugar moieties and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e., β-D-ribonucleosides) or a DNA nucleotide (i.e., β-D-deoxyribonucleoside). Certain embodiments
[00090] Certain embodiments provide methods, compounds and compositions for inhibiting huntingtin expression.
[00091] Certain embodiments provide antisense compounds targeted to a huntingtin nucleic acid. In certain embodiments, the huntingtin nucleic acid is any of the sequences set forth in GENBANK Accession No. NM_002111.6 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_006081.17 truncated from nucleotide 462000 to 634000 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No. NM_010414.1 (incorporated herein as SEQ ID NO: 3), the complement of GENBANK Accession No. NW_001109716.1 truncated at nucleotides 698000 to 866000 ( incorporated herein as SEQ ID NO: 4), and GENBANK Accession No. NM_024357.2 (incorporated herein as SEQ ID NO: 5).
[00092] Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides wherein the linked nucleosides comprise at least 8 contiguous nucleobases of a sequence selected from the nucleobase sequences reported in SEQ ID NO: 6 , 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 35, 36, 10, 11 , 12, 13, 18, 22, 32. In certain embodiments, the modified oligonucleotide comprises at least 9, at least 10, at least 11, or at least 12 contiguous nucleobases of a sequence selected from the nucleobase sequences reported in the SEQ ID No.: 6, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 35, 36, 10, 11, 12, 13, 18, 22, 32. In certain embodiments, the nucleobase sequences are those reported in SEQ ID NOs: 24, 25, 26, 6, 12, 28, 21, 22 , 32, 13. In certain embodiments, the modified oligonucleotide comprises at least 9, at least 10, at least 11 or at least 12 contiguous nucleobases of a sequence selected from the nucleobase sequences reported in SEQ ID NOs: 12, 22, 28, 30, 32 and 33.
[00093] Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 15 to 25 linked nucleosides wherein the linked nucleosides comprise at least 8 contiguous nucleobases of a sequence selected from the nucleobase sequences reported in SEQ ID NO: 6 , 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 35, 36, 10, 11 , 12, 13, 18, 22, 32. In certain embodiments, the modified oligonucleotide comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 contiguous nucleobases of a sequence selected from the nucleobase sequences reported in SEQ ID NOs: 6, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 35, 36, 10, 11, 12, 13, 18, 22, 32. In certain embodiments, the nucleobase sequences are those reported in SEQ ID NOs: 24, 25 , 26, 6, 12, 28, 21, 22, 32, 13. In certain forms of embodiment, the modified oligonucleotide comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 contiguous nucleobases of a sequence selected from the nucleobase sequences reported in SEQ ID NO. °: 12, 22, 28, 30, 32 and 33.
[00094] Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 linked nucleosides wherein the linked nucleosides comprise at least 8 contiguous nucleobases of a sequence selected from the nucleobase sequences reported in SEQ ID NO: 6 , 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 35, 36, 10, 11 , 12, 13, 18, 22 and 32. In certain embodiments, the modified oligonucleotide comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least at least 16, at least 17, or at least 18 contiguous nucleobases of a sequence selected from the nucleobase sequences reported in SEQ ID NO: 6, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 35, 36, 10, 11, 12, 13, 18, 22 and 32. In certain embodiments, the nucleobase sequences are those reported in SEQ ID NOs: 24, 25, 2 6, 6, 12, 28, 21, 22, 32, 13. In certain embodiments, the modified oligonucleotide comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17 or at least 18 contiguous nucleobases of a sequence selected from the nucleobase sequences reported in SEQ ID NOs: 12, 22, 28, 30, 32 and 33.
[00095] Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides wherein the linked nucleosides comprise at least a portion of 8 continuous nucleobases that are complementary to the selected region of nucleotides 4384-4403, 4605 -4624, 4607-4626, 4608-4627, 4609-4628, 4610'4629, 4617-4636, 4622-4639, 4813-4832, 4814-4833, 4823-4842, 4860-4877, 4868-4887, 4925-4944 , 4928-4947, 4931-4950, 4931'4948, 4955-4974, 4960-4977, 5801-5820, 5809-5828, 5809-5826, 101088-101105, 115066-115085, 4607-4626, 4608-4627, 4609 -4628, 4610-4629, 4813-4832, 4862-4881, 5809-5828, 4928-4947 of SEQ ID NO: 1. In certain embodiments the region is selected from 4384, 4403, 4609-4628, 4610- 4629, 4860-4877, 4862-4881, 4925-4944, 4928-4947, 4931-4950, 4955-4974 and 5809-5828 of SEQ ID NO: 1. In certain embodiments the region is selected from 4862-4881 , 4609-4628, 5809-5828, 5809-5826, 5801-5820 and 4955-4974. In certain embodiments, the modified oligonucleotide has a portion of at least 9, at least 10, at least 11, or at least 12 contiguous nucleobase portions that is complementary within a region described herein.
[00096] Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 15 to 25 linked nucleosides wherein the linked nucleosides comprise at least a portion of 8 continuous nucleobases that are complementary to the selected region of nucleotides 4384-4403, 4609 -4628, 4610-4629, 4860-4877, 4862-4881, 4925, 4944, 4928-4947, 4931-4950, 4955-4974 and 5809-5829 of SEQ ID NO: 1. In certain embodiments, the oligonucleotide modified has a portion of at least 9, at least 10, at least 11, at least 12, at least 13, or at least 15 contiguous nucleobase portions that is complementary within a region described herein.
[00097] Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 15 to 25 linked nucleosides wherein the linked nucleosides comprise at least a portion of 8 continuous nucleobases that are complementary to the selected region of nucleotides 4862-4881, 4609 -4628, 5809-5828, 5809-5826, 5801-5820 and 4955'4974. In certain embodiments, the modified oligonucleotide has portions of at least 9, at least 10, at least 11, at least 12, at least 13, or at least 15 of contiguous nucleobases that are complementary within a region described herein.
[00098] Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 linked nucleosides wherein the linked nucleosides comprise at least a portion of 8 continuous nucleobases that are complementary to the selected region of nucleotides 4384-4403, 4609 -4628, 4610-4629, 4860-4877, 4862-4881, 4925, 4944, 4928-4947, 4931-4950, 4955-4974 and 5809-5829 of SEQ ID NO: 1. In certain embodiments, the oligonucleotide modified has portions of at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 contiguous nucleobase portions that is complementary within a region described here.
[00099] Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 linked nucleosides wherein the linked nucleosides comprise at least a portion of 8 continuous nucleobases that are complementary to the selected region of nucleotides 4862-4881, 4609 -4628, 5809-5828, 5809-5826, 5801-5820 and 4955'4974. In certain embodiments, the modified oligonucleotide has portions of at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 contiguous nucleobases that is complementary within a region described herein.
[000100] In certain embodiments, the modified oligonucleotide consists of a single-stranded modified oligonucleotide.
[000101] In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides.
[000102] In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 90% complementary over its full length to a 'nucleobase sequence of SEQ ID NO: 1, 2, 3, 4 or 5. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 95% complementary over its full length to a nucleobase sequence of SEQ ID NO: 1, 2, 3, 4 or 5. In certain embodiments, the modified oligonucleotide is at least 99% complementary in its full length to SEQ ID NO: 1, 2, 3, 4 or 5. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is 100% complementary in its full length to a nucleobase sequence of SEQ ID NO: 1, 2, 3, 4 or 5.
[000103] In certain embodiments, the compound has at least one modified internucleoside linkage. In certain embodiments, the internucleoside linkage is a phosphorothioate internucleoside linkage.
[000104] In certain embodiments, the compound has at least one nucleoside that comprises a modified sugar. In certain embodiments, the at least one modified sugar is a bicyclic sugar. In certain embodiments, the at least one bicyclic sugar comprises a 4'-CH(CH3)-O-2' bridge. In certain embodiments, the at least one modified sugar comprises 2'-O-methoxyethyl.
[000105] In certain embodiments, the compound comprises at least one tetrahydropyran-modified nucleoside in which a tetrahydropyran ring replaces the furanose ring. In certain embodiments, the at least one tetrahydropyran-modified nucleoside has the structure:

[000106] where Bx is an optionally protected heterocyclic base portion. In certain embodiments, the compound has at least one nucleoside that comprises a modified nucleobase. In certain embodiments, the modified nucleobase is a 5-methylcytosine.
[000107] In certain embodiments, the modified oligonucleotide of the compound comprises: (i) a slack segment consisting of linked deoxynucleotides; (ii) a 5' wing segment consisting of linked nucleosides; (iii) a 3' wing segment consisting of linked nucleosides, wherein the slack segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a sugar modified.
[000108] In certain embodiments, the modified oligonucleotide of the compound comprises: (i) a slack segment consisting of ten linked deoxynucleotides; (ii) a 5' wing segment consisting of five linked nucleosides; (iii) a 3' wing segment consisting of five linked nucleosides, wherein the slack segment is positioned immediately adjacent to and between the 5' wing segment and the 3' wing segment, where each nucleoside of each wing segment asa comprises a 2'-O-methoxyethyl sugar and where each internucleoside linkage is a phosphorothioate linkage.
[000109] In certain embodiments, the modified oligonucleotide of the compound comprises: (i) a slack segment consisting of eight linked deoxynucleotides; (ii) a 5' wing segment consisting of six linked nucleosides; (iii) a 3' wing segment consisting of six linked nucleosides, wherein the slack segment is positioned immediately adjacent to and between the 5' wing segment and the 3' wing segment, where each nucleoside of each wing segment asa comprises a 2'-O-methoxyethyl sugar and where each internucleoside linkage is a phosphorothioate linkage.
[000110] In certain embodiments, the modified oligonucleotide of the compound comprises: (i) a slack segment consisting of eight linked deoxynucleotides; (ii) a 5' wing segment consisting of five linked nucleosides; (iii) a 3' wing segment consisting of five linked nucleosides, wherein the slack segment is positioned immediately adjacent to and between the 5' wing segment and the 3' wing segment, where each nucleoside of each wing segment asa comprises a 2'-O-methoxyethyl sugar and where each internucleoside linkage is a phosphorothioate linkage.
[000111] Certain embodiments provide a composition comprising a compound as described herein or a salt thereof and a pharmaceutically acceptable carrier or diluent. In certain embodiments, the composition comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases from a nucleobase sequence selected from the nucleobase sequences reported in SEQ ID NOs. : 6, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 35, 36, 10 , 11, 12, 13, 18, 22 and 32 or a salt thereof and a pharmaceutically acceptable carrier or diluent.
Certain embodiments provide a composition comprising a compound as described herein or a salt thereof and a pharmaceutically acceptable carrier or diluent. In certain embodiments, the composition comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases from a nucleobase sequence selected from the nucleobase sequences reported in SEQ ID NOs. : 12, 22, 28, 30, 32 and 33 or a salt thereof and a pharmaceutically acceptable carrier or diluent.
[000113] Certain embodiments provide methods of treating, preventing or ameliorating Huntington's disease.
[000114] Certain embodiments provide methods comprising administering to an animal a compound as described herein to an animal. In certain embodiments, the method comprises administering to an animal a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence selected from the nucleobase sequences reported in the SEQ ID NO: 6, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 35 , 36, 10, 11, 12, 13, 18, 22 and 32.
[000115] Certain embodiments provide methods comprising administering to an animal a compound as described herein to an animal. In certain embodiments, the method comprises administering to an animal a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence selected from the nucleobase sequences reported in the SEQ ID NO:12, 22, 28, 30, 32 and 33.
[000116] In certain embodiments, the animal is a human.
[000117] In certain embodiments, administration prevents, treats, ameliorates or slows the progression of Huntington's disease as described herein.
[000118] In certain embodiments, the compound is co-administered with a second agent.
[000119] In certain embodiments, the compound and the second agent are administered concurrently.
[000120] In certain embodiments, administration is parenteral administration. In certain embodiments, parenteral administration is intracranial administration. In certain embodiments, intracranial administration is intrathecal or intracerebroventricular administration.
[000121] Certain embodiments further provide a method for reducing huntingtin mRNA or protein expression in an animal which comprises administering to the animal a compound or composition as described herein to reduce huntingtin mRNA or protein expression in the animal. In certain embodiments, the animal is a human. In certain embodiments, the reduction of huntingtin mRNA or protein expression prevents, treats, ameliorates, or slows down the progression of Huntington's disease.
Certain embodiments provide a method of treating a human with Huntington's disease which comprises identifying the human with the disease and administering to the human a therapeutically effective amount of a compound or composition as described herein. In certain embodiments, the treatment reduces a symptom selected from the group consisting of agitation, loss of coordination, unintended initiated movements, unintended incomplete movements, unsteady gait, chorea, rigidity, convulsive movements, abnormal posture, instability, expressions abnormal facial features, difficulty chewing, difficulty swallowing, difficulty speaking, seizure, sleep disturbances, impaired planning, impaired flexibility, impaired abstract thinking, impaired rule acquisition, impaired initiation of appropriate actions, impaired inhibition of inappropriate actions, impaired memory impaired short term, impaired long-term memory, paranoia, disorientation, confusion, hallucination, dementia, anxiety, depression, weakened affect, egocentricity, aggression, compulsive behavior, irritability, suicidal conception, reduced brain mass, muscle atrophy, heart failure, tolerance to impaired glucose, loss of and weight, osteoporosis and testicular atrophy.
[000123] Further provided is a method of reducing or preventing Huntington's disease which comprises administering to human, a therapeutically effective amount of compound or composition as described herein, thereby reducing or preventing Huntington's disease.
[000124] Further provided is a method for ameliorating a symptom of Huntington's disease, which comprises administering to a human in need thereof a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein said oligonucleotide modified specifically hybridizes to SEQ ID NOs: 1, 2, 3, 4 or 5, thereby ameliorating a human Huntington's disease system.
[000125] Further provided is a method for reducing the pathway of progression of a symptom associated with Huntington's Disease, which comprises administering to a human in need thereof a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides , wherein said modified oligonucleotide specifically hybridizes to SEQ ID NOs: 1, 2, 3, 4 or 5, thereby reducing the rate of progression of a Huntington's disease symptom in humans.
[000126] Further provided is a method to reverse degeneration indicated by a symptom associated with Huntington's disease, administering to a human in need thereof, a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein said modified oligonucleotide specifically hybridizes to SEQ ID NOs: 1, 2, 3, 4 or 5, thereby reversing the degeneration indicated by a symptom of Huntington's disease in humans.
[000127] In certain embodiments, the symptom is a physical, cognitive, psychiatric, or peripheral symptom. In certain embodiments, the symptom is a physical symptom selected from the group consisting of agitation, loss of coordination, unintended initiated movements, unintended incomplete movements, unsteady gait, chorea, stiffness, convulsive movements, abnormal posture, instability, abnormal facial expressions, difficulty chewing, difficulty swallowing, difficulty speaking, seizure and sleep disturbance. In certain embodiments, the symptom is a cognitive symptom selected from the group consisting of impaired planning, impaired flexibility, impaired abstract thinking, impaired rule acquisition, impaired onset of appropriate actions, impaired inhibition of inappropriate actions, impaired short-term memory , impaired long-term memory, paranoia, disorientation, confusion, hallucination and dementia. In certain embodiments, the symptom is a psychiatric symptom selected from the group consisting of anxiety, depression, impaired affect, self-centeredness, aggression, compulsive behavior, irritability, and suicidal conception. In certain embodiments, the symptom is a peripheral symptom selected from the group consisting of reduced brain mass, muscle atrophy, heart failure, impaired glucose tolerance, weight loss, osteoporosis, and testicular atrophy.
[000128] Also provided are methods and compounds for the preparation of a medicament for the treatment, prevention or amelioration of Huntington's disease.
[000129] Certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for the treatment, amelioration or prevention of Huntington's disease.
[000130] Certain embodiments provide a compound as described herein for use in treating, preventing or ameliorating Huntington's disease as described herein by therapy and combination with an additional agent or therapy as described herein. Agents or therapies can be co-administered or co-administered.
Certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for the treatment, prevention or amelioration of Huntington's disease as described herein by therapy and combination with an additional agent or therapy as described herein. Agents or therapies can be co-administered or co-administered.
[000132] Certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for the treatment, prevention or amelioration of Huntington's disease as described herein in a patient who is subsequently administered an additional agent or therapy as described herein .
[000133] Certain embodiments provide a kit for the treatment, prevention or amelioration of Huntington's disease as described herein wherein the kit comprises: (i) a compound as described herein and alternatively (ii) an additional agent or therapy such as described here.
[000134] A kit as described which may further include instructions for using a kit to treat, prevent or ameliorate Huntington's disease as described herein by therapy and combination as described herein.
[000135] Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the linked nucleosides comprise at least 8, at least 9, at least 10, at least 11, at least 12, at least at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 contiguous nucleobases of a sequence reported in SEQ ID NO: 6, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 35 or 36, for use in treating an animal having a disease or condition associated with huntingtin by administering to the animal a therapeutically effective amount of the compound such that the expression of huntingtin is inhibited. In certain embodiments, the disease or condition is a neurological disorder. In certain embodiments, the disease or condition is Huntington's Disease. In certain embodiments, the animal is a human.
[000136] Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the linked nucleosides comprise at least 8, at least 9, at least 10, at least 11, at least 12, at least at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 contiguous nucleobases of a sequence reported in SEQ ID NO: 6, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 35 or 36, for use on an animal having a disease or condition associated with huntingtin by administering to the animal a therapeutically effective amount of the compound to prevent, treat, ameliorate or slow the progression of Huntington's disease.
[000137] Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the linked nucleosides comprise at least 8, at least 9, at least 10, at least 11, at least 12, at least at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 contiguous nucleobases of a sequence reported in SEQ ID NO: 12, 22, 28, 30, 32 or 33, for use in an animal having a disease or condition associated with huntingtin by administering to the animal a therapeutically effective amount of the compound such that the expression of huntingtin is inhibited. In certain embodiments, the disease or condition is a neurological disorder. In certain embodiments, the disease or condition is Huntington's Disease.
[000138] In certain embodiments, the animal is a human.
[000139] Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the linked nucleosides comprise at least 8, at least 9, at least 10, at least 11, at least 12, at least at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 contiguous nucleobases of a sequence reported in SEQ ID NO: 12, 22, 28, 30, 32 or 33, for use in an animal having a disease or condition associated with huntingtin by administering to the animal a therapeutically effective amount of the compound to prevent, treat, ameliorate or slow the progression of Huntington's disease.
[000140] Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the linked nucleosides to at least 8, at least 9, at least 10, at least 11, at least 12, at least at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least at 20 contiguous nucleobase portions complementary to the selected region from nucleotides 4384-4403, 4605-4624, 4607-4626, 4608-4627, 4609-4628, 4610-4629, 4617-4636, 4622-4639, 4813-4832, 4814, 4833, 4823-4842, 4860-4877, 4868-4887, 4925-4944, 4928- 4947, 4931-4950, 4931-4948, 4955-4974, 4960-4977, 5801-5820, 5809¬5828, 5809-5826, 101088-101105, 115066-115085, 4607-4626, 4608¬4627, 4609-4628, 4610-4629, 4813-4832, 4862-4881, 5809-5828 and 4928-4947 of SEQ ID NO: 1, for use in an animal having a disease or condition associated with huntingtin by administering to the animal a therapeutically amount. effective of the compound such that huntingtin expression is inhibited.
[000141] Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the linked nucleosides comprise at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least at 20 contiguous nucleobase portions complementary to the selected region from nucleotides 4384-4403, 4605-4624 , 4607-4626, 4608-4627, 4609-4628, 4610-4629, 4617-4636, 4622'4639, 4813-4832, 4814-4833, 4823-4842, 4860-4877, 4868-4887, 4925-4944, 4928 -4947, 4931-4950, 4931-4948, 4955-4974, 4960¬4977, 5801-5820, 5809-5828, 5809-5826, 101088-101105, 115066¬ 115085, 4607-4626, 4608-4627, 4609-4628 , 4610-4629, 4813-4832, 4862-4881, 5809-5828 and 4928-4947 of SEQ ID NO: 1, for use in an animal having a disease or condition associated with huntingtin by administering to the animal an amount therapeutically and effective of the compound in preventing, treating, ameliorating or slowing the progression of Huntington's disease. Antisense Compounds
[000142] Oligomeric compounds, but not limited to, oligonucleotides, oligonucleotides, oligonucleotide analogues, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides and siRNAs. An oligomeric compound can be "antisense" to a target nucleic acid, meaning that it is capable of hybridizing to a target nucleic acid through hydrogen bonding.
[000143] In certain embodiments, an antisense compound has a nucleobase sequence which, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence which, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
[000144] In certain embodiments, an antisense compound targeted to a huntingtin nucleic acid is 12 to 30 nucleotides in length. In other words, antisense compounds are 12 to 30 nucleobases linked. In other embodiments, the antisense compound comprises a modified oligonucleotide consisting of 8 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22 or 20 nucleobases linked. In certain such embodiments, the antisense compound comprises a modified oligonucleotide consisting of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 , 74, 75, 76, 77, 78, 79 or 80 nucleobases linked in length or a range defined by either of the above two values.
[000145] In certain embodiments, the antisense compound comprises a modified truncated or shortened oligonucleotide. The shortened or modified truncated oligonucleotide can have a single nucleoside blunted at the 5' end (5' truncation) or alternatively at the 3' end (3' truncation). A shortened or truncated oligonucleotide can have two voided nucleosides from the 5' end or alternatively it can have two selected subunits from the 3' end. Alternatively, the voided nucleosides can be dispersed throughout the modified oligonucleotide, for example, in an antisense compound having a 5'-end voided nucleoside and a 3'-end voided nucleoside.
[000146] When an additional single nucleoside is present in an elongated oligonucleotide, the additional nucleoside may be located at the 5' or 3' end of the oligonucleotide. When two or more additional nucleosides are present, the added nucleosides can be adjacent to each other, for example, in an oligonucleotide having two nucleosides added at the 5' end (5' addition) or alternatively at the 3' end (3' addition) , of the oligonucleotide. alternatively, the added nucleoside can be dispersed throughout the antisense compound, for example, in an oligonucleotide having a nucleoside added at the 5' end and a subunit added at the 3' end.
[000147] It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide and/or introduce mismatched bases without eliminating the activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13 to 25 nucleobases in length were tested for their ability to induce cleavage of an RNA target in an RNA template. oocyte injection. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 bases mismatched near the ends of the antisense oligonucleotides were able to direct specific cleavage of target mRNA, albeit to a lesser extent than antisense oligonucleotides that did not they contained bad combinations. Similarly, target specific cleavage was achieved using 13 antisense nucleobase oligonucleotides, including those with 1 or 3 mismatches.
[000148] Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) have demonstrated the ability of an oligonucleotide having 100% complementarity to bcl-2 mRNA and having 3 mismatches to bcl-xL mRNA for reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent antitumor activity in vivo.
[000149] Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358,1988) tested a series of 14 tandem antisense nucleobase oligonucleotides and 28 and 42 antisense nucleobase oligonucleotides comprised of the sequence of two or three of tandem antisense oligonucleotides, respectively, for their ability to impede the translation of human DHFR in a rabbit reticulocyte assay. Each of the three antisense nucleobase oligonucleotides 14 alone were able to inhibit, albeit at a more modest level than either the antisense nucleobase oligonucleotides 28 or 42. Reasons for Antisense Compound
[000150] In certain embodiments, antisense compounds targeted to a huntingtin nucleic acid have chemically modified subunits arranged in patterns or motifs to impart antisense properties to the compound, such as enhanced inhibitory activity, affinity for increased binding to a target nucleic acid or resistance to degradation by nucleases in vivo.
[000151] Chimeric antisense compounds typically contain at least one region modified in order to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid and/or increased inhibitory activity. A second region of a chimeric antisense compound can optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.
[000152] Antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an inner region having a plurality of nucleotides that support RNaseH cleavage is positioned between the outer regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the inner region. In the case of an antisense oligonucleotide having a gapmer motif, the slack segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, regions of a gapmer are differentiated by the types of sugar moiety that comprise each distinct region. The types of sugar moiety that are used to differentiate regions of a gapmer may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, modified 2'-nucleosides (such modified 2'-nucleosides may include 2'-MOE and 2'-O-CH3, among others) and modified bicyclic sugar nucleosides (Such bicyclic sugar nucleosides may include those having a bridge of 4'-(CH2)nO-2', where n = 1 or n = 2). Preferably, each distinct region comprises uniform sugar moieties. The wing-slack-wing motif is often described as “XYZ”, where “X” represents the length of the 5' wing region, “Y” represents the length of the slack region, and “Z” represents the length of the wing region 3'. As used herein, a gapmer described as "X-Y-Z" has a configuration such that the clearance segment is positioned immediately adjacent to each of the 5' wing segment and the 3' wing segment. In this way, no intervening nucleotide exists between the 5'-wing segment and the slack segment or the slack segment and the 3'-wing segment. Any of the antisense compounds described herein can have a gapmer motif. In some embodiments X and Z are the same, in other embodiments they are different. In one embodiment, Y is between 8 and 15 nucleotides. X, Y or Z can be any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides. Thus, gapmers include, but are not limited to, for example 5-10-5, 4-8-4, 4-12 to 3, 4¬12 to 4, 3-14-3, 2-13- 5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 6¬8-6 or 5-8-5.
[000153] In certain embodiments, the antisense compound as a "wingmer" motif, having a wing-slack or wing-slack configuration, i.e. an X-Y or Y-Z configuration as described above for the gapmer configuration. Thus, wingmer settings include, but are not limited to, for example, 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3 , 2-10, 1-10, 8-2, 2-13 or 5-13.
In certain embodiments, antisense compounds targeted to a huntingtin nucleic acid possess a 5-10-5 degapmer motif.
In certain embodiments, antisense compounds targeted to a huntingtin nucleic acid possess a 6-8-6 gapmer motif.
[000156] In certain embodiments, antisense compounds targeted to a huntingtin nucleic acid possess a 5-8-5 gapmer motif.
[000157] In certain embodiments, an antisense compound targeted to a huntingtin nucleic acid has an extended slack motif.
[000158] In certain embodiments, an antisense slack amplified oligonucleotide targeted to a huntingtin nucleic acid has a slack segment of ten 2'-deoxyribonucleotides positioned immediately adjacent to and between the five chemically modified nucleoside wing segments. In certain embodiments, the chemical modification comprises a 2'-sugar modification. In another embodiment, the chemical modification comprises a 2'-MOE sugar modification.
[000159] In certain embodiments, an antisense slack amplified oligonucleotide targeted to a huntingtin nucleic acid has an eight 2'-deoxyribonucleotide slack segment positioned immediately adjacent to and between the chemically modified five nucleoside wing segments. In certain embodiments, the chemical modification comprises a 2'-sugar modification. In another embodiment, the chemical modification comprises a 2'-MOE sugar modification.
[000160] In certain embodiments, an antisense slack amplified oligonucleotide targeted to a huntingtin nucleic acid has an eight 2'-deoxyribonucleotide slack segment positioned immediately adjacent to and between the chemically modified six nucleoside wing segments. In certain embodiments, the chemical modification comprises a 2'-sugar modification. In another embodiment, the chemical modification comprises the 2'-MOE sugar modification.
[000161] Target Nucleic Acids, Target Regions and Nucleotide Sequences
[000162] Huntingtin encoding nucleotide sequences include, without limitation, the following: GENBANK Accession No. NM_002111.6, first deposited with GENBANK® on May 31, 2006 incorporated herein as SEQ ID NO: 1; GENBANK Accession No. NT_006081.17 truncated from nucleotides 462000 to 634000, first deposited with GENBANK® on August 19, 2004 and incorporated herein as SEQ ID NO:2; GENBANK Accession No. NM_010414.1, first filed with GENBANK® on March 23, 2004, incorporated herein as SEQ ID NO: 3; the complement of GENBANK Accession No. NW_001109716.1 truncated at nucleotides 698000 to 866000, first deposited with GENBANK® on June 14, 2006, incorporated herein as SEQ ID No.: 4 and GENBANK Accession No. NM_024357.2, first deposited with GENBANK® on June 5, 2008, incorporated herein as SEQ ID NO: 5.
[000163] It is understood that the sequence shown in each SEQ ID NO: in the examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage or a nucleobase. As such, the antisense compounds defined by a SEQ ID NO: may independently comprise one or more modifications to a sugar moiety, an internucleoside linkage or a nucleobase. The antisense compounds described by Isis Number (Isis No.) indicates a combination of nucleobase sequence and motif.
[000164] In certain embodiments, the target region is a structurally defined region of the target nucleic acid. For example, the target region can span the 3' UTR, 5' UTR, an exon, an intron, an exon/intron junction, a coding region, a translation start region, a translation end region, or other defined nucleic acid region. Structurally defined regions for huntingtin can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference. In certain embodiments, the target region may span the sequence from a 5' target site of one target segment within the target region to a 3' target site of another target segment within the target region.
[000165] Targeting includes determining at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in mRNA target nucleic acid levels. In certain embodiments, the desired effect is a reduction in protein levels encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.
[000166] The target region can contain one or more target segments. Multiple target segments within a target region can be overlapping. Alternatively, these can be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain embodiments, target segments within the target region are separated by several nucleotides i.e. is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 nucleotides in the target nucleic acid or is in a range defined by any two of the preceding values. In certain embodiments, target segments within the target region are separated by no more than or no more than about 5 nucleotides in the target nucleic acid. In certain embodiments, target segments are contiguous. Target regions are defined by a lane having a starting nucleic acid that is any of the 5' target sites or 3' target sites listed herein.
[000167] Suitable target segments can be found within a 5' UTR, a coding region, a 3' UTR, an intron, an exon, or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment can specifically exclude a certain structurally defined region, such as the start codon or stop codon.
[000168] The determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm can be used to identify regions of similarity between different nucleic acids. This comparison can avoid the selection of antisense compound sequences that can hybridize in a non-specific manner to sequences other than a selected nucleic acid (i.e., non-target or non-target sequences).
[000169] There may be variation in the activity (eg, as defined by percent reduction in target nucleic acid levels) of antisense compounds within an active target region. In certain embodiments, reductions in huntingtin mRNA levels are indicative of inhibition of huntingtin expression. Reductions in huntingtin protein levels are also indicative of inhibition of target mRNA expression. Furthermore, phenotypic changes are indicative of inhibition of huntingtin expression expression. For example, an increase in brain size to normal, improvement in motor coordination, decrease in ongoing muscle spasms (dystonia), decrease in irritability and/or anxiety, improvement in memory or an increase in energy, among other phenotypic changes that may be experienced. the rehearsal. Other phenotypic indications, eg symptoms associated with Huntington's disease, can also be estimated as described below. Hybridization
[000170] In some embodiments, hybridization occurs between an antisense compound disclosed herein and a huntingtin nucleic acid. The most common hybridization mechanism involves hydrogen bonding (eg, Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonding) between the complementary nucleobases of the nucleic acid molecules.
[000171] Hybridization can occur under varying conditions. Stringent conditions are sequence dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.
[000172] Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable to a huntingtin nucleic acid. Complementarity
[000173] An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can bind hydrogen with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (by example, antisense inhibition of a target nucleic acid such as huntingtin nucleic acid).
[000174] An antisense compound can hybridize to one or more segments of a huntingtin nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (eg, an arc structure, structure at odds or hairpin).
[000175] In certain embodiments, the provided antisense compounds, or a specified portion thereof, are or are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity to a huntingtin nucleic acid, a target region, target segment or its specified portion. The percent complementarity of an antisense compound to a target nucleic acid can be determined using routine methods.
[000176] For example, an antisense compound in which 18 of the 20 nucleobases of the antisense compound are complementarity to a target region and must therefore specifically hybridize, must represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases can be grouped or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound that is 18 nucleobases in length having 4 (four) non-complementary nucleobases that are flanked by two complete complementarity to the target nucleic acid should have 77.8% total complementarity to the target nucleic acid and, therefore, should be within the scope of the present invention. The percent complementarity of an antisense compound with a target nucleic acid region can be routinely determined using BLAST programs (local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol ., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity can be determined, for example, by the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482 489).
[000177] In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e., 100% complementarity) to a target nucleic acid or specified portion thereof. For example, the antisense compound can be fully complementary to a huntingtin nucleic acid or target region or a target segment or target sequence thereof. As used herein, "fully complementary" means that each nucleobase of an antisense compound is capable of precise base pair formation with the corresponding nucleobases of a target nucleic acid. For example, 20 nucleobase antisense compounds is fully complementary to a target sequence that is 400 nucleobases in length, as long as a corresponding 20 nucleobase portion of the target nucleic acid is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be "fully complementary" to a target sequence that is 400 nucleobases in length. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion where each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the full-length 30-nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on how the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.
[000178] The site of a non-complementary nucleobase may be at the 5' end or 3' end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be in an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they can be contiguous (i.e., linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located on the wing segment of the antisense gapmer oligonucleotide.
[000179] In certain embodiments, antisense compounds that are or are not and up to 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleobases in length comprise no more than 4, not no more than 3, no more than 2 or no more than 1 non-complementary nucleobase(s) with respect to a target nucleic acid, such as a huntingtin nucleic acid or specified portion thereof.
[000180] In certain embodiments, antisense compounds that are of or that are up to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2 or no more than 1 nucleobase( s) non-complementary(s) with respect to a target nucleic acid, such as huntingtin nucleic acid or its specified portion.
[000181] Antisense compounds provided herein also include those that are complementary to a portion of a target nucleic acid. As used herein, "portion" refers to a defined number of contiguous (i.e., linked) nucleobases within a region or segment of a target nucleic acid. A "portion" can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds are complementary to at least an 8-nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least 12 nucleobase portions of a target segment. In certain embodiments, antisense compounds are complementary to at least 15 nucleobase portions of a target segment. Also considered are antisense compounds that are complementary to at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleobase portions of a target segment or a band defined by any two of these values. Identity
[000182] The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO: or compound represented by a specific Isis number or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed therein if it has the same nucleobase pair formation capability. For example, an RNA that contains uracil in place of thymidine in a disclosed DNA sequence should be considered identical to the DNA sequence since both uracil and thymidine are paired with adenine. Shortened or extended versions of the antisense compounds described herein, as well as compounds having non-identical bases relative to the antisense compounds provided herein are contemplated. Non-identical bases can be adjacent to each other or dispersed throughout the antisense compound. The percent identity of an antisense compound is calculated according to a number of bases that have identical base pair formation with respect to the sequence to which it is being compared.
[000183] In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compound or SEQ ID NO. or a portion thereof, disclosed herein. Modifications
[000184] A nucleoside is a base sugar combination. The nucleobase (also known as the base) portion of the nucleotide is usually a heterocyclic base portion. Nucleotides are nucleosides that further include a phosphate group covalently attached to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group may be attached to the 2', 3' or 5' hydroxyl portion of the sugar. Oligonucleotides are formed by covalently linking adjacent nucleosides to one another to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, phosphate groups are commonly referred to as forming an oligonucleotide internucleoside bond.
[000185] Modifications to antisense compounds encompass substitutions or changes to internucleoside bonds, sugar moieties or nucleobases. Modified antisense compounds are often preferred in natural forms because of desirable properties, such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acid, increased stability in the presence of nucleases, or increased inhibitory activity.
[000186] Chemically modified nucleosides can also be used to increase the binding affinity of a shortened or truncated antisense oligonucleotide to its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides. Modified internucleoside linkages
[000187] The naturally occurring internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage. Antisense compounds having one or more modified-occurring, i.e., non-naturally occurring internucleoside linkages are often selected from antisense compounds having naturally-occurring internucleoside linkages because of desirable properties, such as, for example, cellular uptake enhanced, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
[000188] Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom, as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate and phosphorothioates. Methods of preparing phosphorus-containing and non-phosphorus-containing bonds are also known.
[000189] In certain embodiments, antisense compounds targeted to a huntingtin nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage. Modified Sugar Servings
[000190] Antisense compounds may optionally contain one or more nucleosides in which the sugar group has been modified. Such a modified sugar nucleoside may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include, without limitation, addition of substituent groups (including 5' and 2' substituent groups, bridging non-geminal ring atoms to form bicyclic nucleic acids (BNA), oxygen atom replacement of the ribosyl ring with S, N(R) or C(R1)(R)2 (R = H, C1-C12 alkyl or a protecting group) and combinations thereof Examples of chemically modified sugars include nucleoside substituted by 2' -F-5'-methyl (see PCT International Application WO 2008/101157 published on 8/21/08 for other 5',2'-bis substituted nucleosides disclosed) or replacement of the ribosyl ring oxygen atom with S with the additional substitution at the 2' position (see Published US Patent Application US2005-0130923, published June 16, 2005) or alternatively 5'-replacement of a BNA (see International PCT Application WO 2007/134181 Published 11/22/ 07 where LNA is substituted with for example a 5'-methyl group or a 5'-vinyl group).
[000191] Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3 and 2 substituent groups '-O(CH2)2OCH3. The substituent at the 2' position can also be selected from allyl, amino, azido, thio, O-allyl, O-C1-C10 alkyl, OCF3, O(CH2)2SCH3, O(CH2)2-ON(Rm)(Rn ) and O-CH 2 -C(=O)-N(Rm)(Rn), where each Rm and Rn is independently substituted or unsubstituted C1¬C10 alkyl.
[000192] Examples of bicyclic nucleic acids (BNAs) include without limitation nucleosides that comprise a bridge between the 4' and the 2' ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more BNA nucleosides wherein the bridge comprises one of the formulas: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)-O-2' (LNA); 4'-(CH2)2-O-2' (ENA); 4'-C(CH3)2-O-2' (see PCT/US2008/068922); 4'-CH(CH3)--O-2' and 4'-C-H(CH2OCH3)--O-2' (see U.S. Patent 7,399,845, filed July 15, 2008); 4'-CH2-N(OCH3)-2' (see PCT/US2008/064591); 4'-CH2-O-N(CH3)-2' (see Published U.S. Patent Application US2004-0171570, published September 2, 2004); 4'-CH2-N(R)-O-2' (see U.S. Patent 7,427,672, filed September 23, 2008); 4'-CH2-C(CH3)-2' and 4'-CH2-C-(=CH2)-2' (see PCT/US2008/066154) and where R is independently H, C1-C12 alkyl or a protection group. Each of the foregoing BNAs include various stereochemical sugar configurations including, for example, α-L-ribofuranose and β-D-ribofuranose (see PCT International Application PCT/DK98/00393, published March 25, 1999 as WO 99/14226 ).
[000193] In certain embodiments, nucleosides are modified by replacing the ribosyl ring with a sugar substitute. Such modification includes, without limitation, replacement of the ribosyl ring with a surrogate ring system (sometimes referred to as DNA analogs) such as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring such as a having one of the formula:

[000194] Many other bicycle and tricycle sugar ring system substitutes are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see, for example, abstract article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854). Such ring systems can undergo several additional replacements to enhance activity.
[000195] Methods for the preparations of the modified sugars are well known to that person skilled in the art.
[000196] In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are held for hybridization to an appropriate nucleic acid target.
[000197] In certain embodiments, antisense compounds targeted to a huntingtin nucleic acid comprise one or more nucleotides having the modified sugar moieties. In certain embodiments, the modified sugar moiety is 2'-MOE. In certain embodiments, the modified 2'-MOE nucleotides are arranged in a gapmer motif. Modified Nucleobase
[000198] Nucleobase (or base) modifications or substitutions are structurally distinguishable from already functionally interchangeable with naturally occurring or modified synthetic nucleobase. Both natural and modified nucleobase are able to participate in hydrogen bonding. Such nucleobase modifications may impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobase includes synthetic or natural nucleobases such as, for example, 5-methylcytokine (5-me-C). Certain nucleobase substitutions, including 5-methylcytokine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytokine substitutions have been shown to increase the stability of the nucleic acid duplex by 0.6 to 1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., Antisense Research and Applications , CRC Press, Boca Raton, 1993, pp. 276-278).
[000199] Additional unmodified nucleobase includes 5-hydroxymethyl cytokine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytokine, 5-halouracil and cytokine, 5-propynyl (-C=C-CH3) uracil and cytokine and other alkynyl derivatives of the bases of pyrimidine, 6-azo uracil, cytokine and thymine, 5 -uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytokines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3- deazaadenine.
[000200] Heterocyclic base moieties can also include where the purine or pyrimidine base is substituted with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include substituted 5-pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5 -propynylcytocin.
[000201] In certain embodiments, antisense compounds targeted to huntingtin nucleic acid comprise one or more modified nucleobase. In certain embodiments, clearance-amplified antisense oligonucleotides targeted to huntingtin nucleic acid comprise one or more modified nucleobase. In certain embodiments, the modified nucleobase is 5-methylcytokine. In certain embodiments, each cytokine is a 5-methylcytosine.
[000202] Compositions and methods for formulating pharmaceutical compositions
[000203] Antisense oligonucleotides can be mixed with pharmaceutically acceptable inert or active substance for the preparation of pharmaceutical compositions or formulations. Compositions and methods for formulating the pharmaceutical compositions are dependent on various criteria, including, but not limited to, the route of administration, extent of disease or dosage to be administered.
The antisense compound targeted to huntingtin nucleic acid can be used in pharmaceutical compositions by combining the antisense compound with a pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate buffered saline (PBS). PBS is a suitable diluent for use in compositions to be released parenterally. Accordingly, in one embodiment, used in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to huntingtin nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.
[000205] Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters or salts of such esters or any other oligonucleotide which, on administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
[000206] A prodrug may include the incorporation of additional nucleosides at one or both ends of an antisense compound that are cleaved by endogenous nucleases within the body to form the active antisense compound. Conjugated antisense compounds
[000207] Antisense compounds can be covalently linked to one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugated groups include carbohydrates, phospholipids, biotin, phenazine, folatol, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins and pigments.
[000208] Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached at one or both termini of the antisense compounds to enhance properties such as, for example, nuclease stability. Included in the stabilization groups are roof structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation and may aid delivery and/or localization within the cell. The cover may be present at the 5' end (5' cover) or at the 3' end (3' cover) or it may be present at both ends. Cover structures are well known in the art and include, for example, inverted deoxy basic covers. Further 3' and 5' stabilizing groups that can be used to cover one or both ends of an antisense compound to impart nuclease stability include that disclosed in WO 03/004602 published January 16, 2003. Cell culture and the treatment of antisense compounds
[000209] The effects of antisense compounds on the level, activity or expression of huntingtin nucleic acid can be tested in vitro in a variety of cell types. Cell types for such analyzes are available from commercial vendors (eg, American Type Culture Collection, Manassus, VA; Zen-Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD) and the cells are cultured according to vendors instructions using commercially available reagents (eg, Invitrogen Life Technologies, Carlsbad, CA). Illustrative cell types include, but are not limited to, HepG2 cells, Hep3B cells, primary hepatocytes, A549 cells, GM04281 fibroblasts, and LLC-MK2 cells. In vitro testing of antisense oligonucleotides
[000210] Described herein are methods for treating cells with antisense oligonucleotides, which may be appropriately modified for treatment with other antisense compounds.
[000211] In general, cells are treated with antisense oligonucleotides when cells have reached approximately 60 to 80% confluence in culture.
[000212] A commonly used reagent to introduce antisense oligonucleotides into cultured cells include the LIPOFECTIN® cationic lipid transfection reagent (Invitrogen, Carlsbad, CA). Antisense oligonucleotides are mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, CA) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN® concentration typically ranging from 2 to 12 ug/mL per 100 nM of antisense oligonucleotide.
[000213] Another reagent used to introduce antisense oligonucleotides into cultured cells include LIPOFECTAMINE 2000® (Invitrogen, Carlsbad, CA). Antisense oligonucleotide is mixed with LIPOFECTAMINE 2000® in OPTI-MEM® 1 Reduced Serum Medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE® concentration that typically ranges from 2 to 12 ug/ml per 100 nM antisense oligonucleotide.
[000214] Another reagent used to introduce antisense oligonucleotides into cultured cells include Cytofectin® (Invitrogen, Carlsbad, CA). Antisense oligonucleotide is mixed with Cytofectin® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of antisense oligonucleotide and a Cytofectin® concentration typically ranging from 2 to 12 ug µg/ml per 100 nM antisense oligonucleotide.
[000215] Another technique used to introduce antisense oligonucleotides into cultured cells include electroporation.
[000216] Cells are treated with antisense oligonucleotides by routine methods. Cells are typically harvested 16 to 24 hours after antisense oligonucleotide treatment, in which time RNA or target nucleic acid protein levels are measured by methods known in the art and described herein. In general, when treatments are performed on multiple replicates, the data is presented as the mean of the replicate treatments.
[000217] The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE2000®, Lipofectin or Cytofectin. Antisense oligonucleotides are used at the highest concentrations ranging from 625 to 20,000 nM when transfected using electroporation. RNA isolation
[000218] RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. RNA isolation methods are well known in the art. RNA is prepared using methods well known in the art, for example, using TRIZOL® reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommended protocols. Analysis of inhibition of target levels or expression
[000219] Inhibition of the levels or expression of a huntingtin nucleic acid can be subjected to the assay in a variety of ways known in the art. For example, target nucleic acid levels can be quantified by, for example, Northern blot analysis, competitive polymerase chain reaction (PCR) or quantitative real-time PCR. RNA analysis can be performed on a cellular RNA or poly(A)+ mRNA. RNA isolation methods are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative PCR can be conveniently followed using the commercially available ABI PRISM® 7600, 7700 or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, CA and used according to the manufacturer's instructions. Quantitative real-time PCR analysis of target RNA levels
[000220] Quantitation of target RNA levels can be followed by quantitative real-time PCR using the ABI PRISM® 7600, 7700 or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to the instructions from the manufacturer.
[000221] Quantitative real-time PCR methods are well known in the art.
[000222] Prior to real-time PCR, isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) which is then used as the substrate for real-time PCR amplification. Real-time RT and PCR reactions are sequentially performed in the same sample reservoir. RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, CA). The RT, real-time PCR reactions are performed by methods well known to that person skilled in the art.
[000223] The target amounts of the gene (or RNA) obtained by real-time PCR are normalized using the expression level of a gene whose expression is constant, such as cyclophilin A or by quantifying total RNA using RIBOGREEN® (Invitrogen, Inc .Carlsbad, CA). Cyclophilin A expression is quantified by real-time PCR, as it is performed simultaneously with the target, multiplexed or separately. Total RNA is quantified using the RIBOGREEN® RNA Quantitation Reagent (Invitrogen, Inc. Eugene, OR). Methods of RNA quantification by RIBOGREEN® are taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR® 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN® fluorescence.
[000224] Probes and primers are designed to hybridize huntingtin nucleic acid. Methods for designing real-time PCR probes and primers are well known in the art and can include the use of software such as PRIMER EXPRESS® software (Applied Biosystems, Foster City, CA). Analysis of protein levels
[000225] Antisense inhibition of huntingtin nucleic acid can be assessed by measuring protein levels. Huntingtin protein levels can be assessed or quantified in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays. protein (eg, caspase activity assays), immunohistochemistry, immunocytochemistry, or fluorescence activated cell sorting (FACS). Targeted antibodies can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI) or can be prepared via standard monoclonal or polyclonal antibody generation methods as well. known in the art. Antibodies useful for detecting human and mouse huntingtin are commercially available. In vivo testing of antisense compounds
[000226] Antisense compounds, eg antisense oligonucleotides, are tested in animals to assess their ability to inhibit their expression of huntingtin and produce phenotypic changes. The test can be performed on normal animals or on models of experimental disease. For administration to animals, antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent such as phosphate buffered saline. Administration includes parenteral routes of administration. Following a period of treatment with antisense oligonucleotides, RNA is isolated from the tissue and changes in the nucleic acid of huntingtin expression are measured. Changes in huntingtin protein levels are also measured. certain compounds
[000227] Approximately one thousand seven hundred newly designed antisense compounds of various lengths, motifs and secondary chain composition were tested for their effect on human huntingtin mRNA in vitro in various cell types. The new compounds were compared with about two hundred and fifty previously designed compounds including ISIS 387916 which has previously determined to be one of the most potent antisense compounds in vitro (see for example, US Patent Publication Nos. 2008/0039418 and 2007 /0299027. About seventeen hundred newly designed antisense compounds, about sixty compounds were selected for further study based on in vitro potency compared to ISIS 387916. Selected compounds were tested for systemic tolerability (see Example 3) and activity and tolerability in the brain of BACHD mice (see Example 4) compared to previously designed ISIS 388241 and ISIS 387916. From these studies, compounds having a nucleobase sequence of a sequence reported in SEQ ID NO: 6, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 35, 36, 10, 11, 12, 13, 18, 22 or 32 were selected as having tolerability. high power and high power in vivo. By virtue of their complementary sequence, the compounds are complementary to regions 4384-4403, 4605-4624, 4607-4626, 4608-4627, 4609-4628, 4610-4629, 4617-4636, 4622-4639, 4813-4832, 4814 -4833, 4823-4842, 4860-4877, 4868-4887, 4925-4944, 4928-4947, 4931-4950, 4931-4948, 4955-4974, 4960-4977, 5801-5820, 5809-5828, 5809-5826 , 101088-101105, 115066-115085, 4607-4626, 4608-4627, 4609-4628, 4610-4629, 4813-4832, 4862-4881, 5809-5828 or 4928-4947 of SEQ ID NO: 1. In certain embodiments, compounds targeting the listed regions, further as described herein, comprise a modified oligonucleotide having some nucleobase portion of the sequence reported in SEQ ID No., further as described herein. In certain embodiments, compounds targeting the regions listed or having a nucleobase portion of a sequence reported in SEQ ID NO. listed may be of various lengths, further as described herein, and one may have of various motifs, further as described. in this. In certain embodiments, a compound targeting a region or having a nucleobase portion of a sequence reported in SEQ ID NO. listed has the specific length and motif as indicated by ISIS No.: ISIS 419628, ISIS 419637, ISIS 419640, ISIS 419641, ISIS 451541, ISIS 419642, ISIS 436665, ISIS 436671, ISIS 436684, ISIS 436689, ISIS 436754, ISIS 437168, ISIS 437175, ISIS 437441, ISIS 437442, ISIS 437507, ISIS 437527, ISIS 443139, ISIS 444578, ISIS 444578 , ISIS 444591, ISIS 444607, ISIS 444608, ISIS 444615, ISIS 444618, ISIS 444627, ISIS 444652, ISIS 444658, ISIS 444659, ISIS 444660, ISIS 444661 or ISIS 444663.
[000228] The compounds described above as having high in vivo potency and tolerability were then tested by CNS bolus injection into rat to assess additional neurotoxicity (see Example 5) along with several additional compounds having a nucleobase sequence of a sequence reported in SEQ ID NO: 7, 8, 11, 16, 17. Of these, ten compounds having a nucleobase sequence of a sequence reported in SEQ ID NO: 24, 25, 26, 6, 12, 28, 21, 22, 32 or 13 were selected as having high tolerability. By virtue of their complementary sequence, the compounds are complementary to regions 4384-4403, 4609-4628, 4610-4629, 4860-4877, 4862-4881, 4925-4944, 4928-4947, 4931-4950, 4955-4974 or 5809 -5829 of SEQ ID NO: 1. In certain embodiments, compounds targeting the listed regions, further as described herein, comprise a modified oligonucleotide having some nucleobase portion of the sequence reported in SEQ ID NO. in this. In certain embodiments, compounds targeting the regions listed or having a nucleobase portion of a sequence reported in SEQ ID NO. listed may be of various lengths, further as described herein, and may have one of several motifs, further as described. in this. In certain embodiments, a compound targeting a region or having a nucleobase portion of a sequence reported in SEQ ID NO. listed has the specific length or motif as indicated by ISIS No.: ISIS 419640, ISIS 419641, ISIS 419642, ISIS 436665, ISIS 436671, ISIS 436689, ISIS 437507, ISIS 443139, ISIS 444591 and ISIS 444661. The selected compounds were compared to the previously designed compound ISIS 388241 by ICV administration in the BACHD mice.
[000229] Additional studies were then performed on the compounds described above as having high in vivo potency and tolerability. Additional studies were designed to assess additional neurotoxicity. Studies include ICV administration in wild-type mice (see Example 16) and bolus administration in rats (see Example 17). SEQ ID NOs: 12, 22, 28, 30, 32 and 33 were selected as having the high neurotolerability. By virtue of their complementary sequence, the compounds are complementary to regions 4862-4881, 4609-4628, 5809-5828, 5809-5826, 5801-5820 and 4955-4974 of SEQ ID NO: 1. In certain embodiments, compounds targeting the regions listed, further as described herein, comprise a modified oligonucleotide having the same nucleobase portion of the sequence reported in SEQ ID NOs, further as described herein. In certain embodiments, compounds targeting the regions listed or having a nucleobase portion of a sequence reported in SEQ ID NO. listed may be of various lengths, further as described herein, and may have one of several motifs, further as described in this. In certain embodiments, a compound targeting a region or having a nucleobase portion of a sequence reported in SEQ ID NO. listed has the specific length or motif as indicated by ISIS 388241, ISIS 443139, ISIS 436671, ISIS 444591, ISIS 437527, ISIS 444584, ISIS 444652 and ISIS 436689.
[000230] Consequently, provided in this are antisense compounds with improved characteristics. In certain embodiments, provided herein are compounds comprising an oligonucleotide further modified as described herein targeted or specifically hybridizable to the nucleotide region of SEQ ID NO:1.
[000231] In certain embodiments, compounds as described herein are effective by virtue of having at least an in vitro IC50 of less than 7 uM, less than 6 uM, less than 5, uM, less than 4 uM, less than 3 uM, less than 2 uM, less than 1 uM when released to a human fibroblast cell line as described herein or an ED50 of less than 10 μg, less than 9 μg, less than 8 μg, less than 7.5 μg, less than 7.4 μg, less than 7.0 μg, less than 6 μg, less than 5 μg, less than 4 μg, less than 3 μg or less than 2 μg by bolus injection. As described herein, ICV infusion can result in 3- to 4-fold higher ED50 values for the compounds described herein. In certain embodiments, compounds as described herein are highly tolerable as demonstrated to have at least an increase in ALT or AST value of no more than 4-fold, 3-fold or 2-fold in saline treated animals; an increase in liver, spleen, or kidney weight of no more than 30%, 20%, 15%, 12%, 10%, 5% or 2%; or an increase in AIF1 levels not more than 350%, 300%, 275%, 250% 200%, 150% or 100% in the control. certain indications
[000232] In certain embodiments, methods of treating an individual are provided which comprise administering one or more pharmaceutical compositions as described herein. In certain embodiments, the individual has Huntington's disease.
[000233] As shown in the examples below, huntingtin-targeted compounds as described here have been shown to reduce the severity of physiological symptoms of Huntington's disease. In certain experiments, the reduced rate of degeneration of compounds, for example, animals continued to experience symptoms, but symptoms were less severe compared to untreated animals. In other of the experiments, however, the compounds appeared to result in regeneration of function over time; for example, animals treated for a longer period of time experienced less severe symptoms than those given the compounds for a shorter period of time. As discussed above, Huntington's disease is a degenerative disease with a progression typified by increased severity of symptoms over time. The ability of the compounds exemplified below to restore function, therefore, demonstrates that disease symptoms can be reversed by treatment with a compound as described herein.
[000234] Consequently, methods for ameliorating a symptom associated with Huntington's disease in a patient in need thereof are provided here. In certain embodiments, a method of reducing the rate of onset of a symptom associated with Huntington's disease is provided. In certain embodiments, a method of reducing the severity of a symptom associated with Huntington's disease is provided. In certain embodiments, a method of regenerating neurological function as shown by an amelioration of a symptom associated with Huntington's disease is provided. In such embodiments, the method comprises administering to a subject in need thereof, a therapeutically effective amount of a compound targeted to a huntingtin nucleic acid.
[000235] Huntington's disease is characterized by numerous physical, neurological, psychiatric and/or peripheral symptoms. Any symptoms known to those of skill in the art that will be associated with Huntington's disease can be ameliorated or otherwise modulated as set out above in the methods described above. In certain embodiments, the symptom is a physical symptom selected from the group consisting of agitation, loss of coordination, unintended initiated movements, unintended incomplete movements, unsteady gait, chorea, stiffness, convulsive movements, abnormal posture, instability, abnormal facial expressions, difficulty chewing, difficulty swallowing, difficulty speaking, seizure and sleep disturbance. In certain embodiments, the symptom is a cognitive symptom selected from the group consisting of impaired planning, impaired flexibility, impaired abstract thinking, impaired rule acquisition, impaired onset of appropriate actions, impaired inhibition of inappropriate actions, impaired short-term memory , impaired long-term memory, paranoia, disorientation, confusion, hallucination and dementia. In certain embodiments, the symptom is a psychiatric symptom selected from the group consisting of anxiety, depression, impaired affect, self-centeredness, aggression, compulsive behavior, irritability, and suicidal conception. In certain embodiments, the symptom is a peripheral symptom selected from the group consisting of reduced brain mass, muscle atrophy, heart failure, impaired glucose tolerance, weight loss, osteoporosis, and testicular atrophy.
[000236] In certain embodiments, the symptom is restlessness. In certain embodiments, the symptom is loss of coordination. In certain embodiments, the symptom is unintentional initiated movements. In certain embodiments, the symptom is unintentional incomplete movements. In certain embodiments, the symptom is step-shifting. In certain embodiments, the symptom is chorea. In certain embodiments, the symptom is rigidity. In certain embodiments, the symptom is convulsive movements. In certain embodiments, the symptom is abnormal posture. In certain embodiments, the symptom is instability. In certain embodiments, the symptom is abnormal facial expressions. In certain embodiments, the symptom is difficulty chewing. In certain embodiments, the symptom is difficulty in swallowing. In certain embodiments, the symptom is speech impairment. In certain embodiments, the symptom is seizures. In certain embodiments, the symptom is sleep disturbance.
[000237] In certain embodiments, the symptom is impaired planning. In certain embodiments, the symptom is impaired flexibility. In certain embodiments, the symptom is impaired abstract thinking. In certain embodiments, the symptom is impaired rule acquisition. In certain embodiments, the symptom is impaired onset of appropriate actions. In certain embodiments, the symptom is impaired inhibition of inappropriate actions. In certain embodiments, the symptom is impaired short-term memory. In certain embodiments, the symptom is impaired long-term memory. In certain embodiments, the symptom is paranoia. In certain embodiments, the symptom is disorientation. In certain embodiments, the symptom is confusion. In certain embodiments, the symptom is hallucination. In certain embodiments, the symptom is dementia.
[000238] In certain embodiments, the symptom is anxiety. In certain embodiments, the symptom is depression. In certain embodiments, the symptom is impaired affect. In certain embodiments, the symptom is self-centeredness. In certain embodiments, the symptom is aggression. In certain embodiments, the symptom is compulsive behavior. In certain embodiments, the symptom is irritability. In certain embodiments, the symptom is suicidal conception.
[000239] In certain embodiments, the symptom is reduced brain mass. In certain embodiments, the symptom is muscle atrophy. In certain embodiments, the symptom is heart failure. In certain embodiments, the symptom is impaired glucose tolerance. In certain embodiments, the symptom is weight loss. In certain embodiments, the symptom is osteoporosis. In certain embodiments, the symptom is testicular atrophy.
[000240] In certain embodiments, Huntington's disease symptoms may be quantifiable. For example, osteoporosis can be measured and quantified by, for example, bone density scans. For such symptoms, in certain embodiments, the symptom can be reduced by about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% or a range defined by any two of these values.
In certain embodiments, methods of treating an individual are provided which comprise administering one or more pharmaceutical compositions as described herein. In certain embodiments, the individual has Huntington's disease.
[000242] In certain embodiments, administration of an antisense compound targeted to a huntingtin nucleic acid results in the reduction of huntingtin expression by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% or a range defined by any two of these values.
In certain embodiments, pharmaceutical compositions comprising a huntingtin-targeted antisense compound are used for the preparation of a medicament for the treatment of a patient suffering from or susceptible to Huntington's disease.
[000244] In certain embodiments, the methods described herein include administering a compound comprising a modified oligonucleotide having a portion of contiguous nucleobases as described herein from a sequence reported in SEQ ID NO: 6, 9, 10, 11, 12 , 13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 35, 36, 10, 11, 12, 13, 18 , 22 or 32. In certain embodiments, the methods described herein include administering a compound comprising a modified oligonucleotide having a portion of contiguous nucleobases as described herein from a sequence reported in SEQ ID NOs: 12, 22, 28, 30 , 32 and 33. Administration
[000245] In certain embodiments, compounds and compositions as described herein are administered parenterally.
[000246] In certain embodiments, parenteral administration occurs by infusion. The infusion can be chronic or continuous or short or intermittent. In certain embodiments, pharmaceutical agents to be infused are delivered with a pump. In certain embodiments, parenteral administration is by injection.
[000247] In certain embodiments, compounds and compositions are released to the CNS. In certain embodiments, compounds and compositions are released into the cerebrospinal fluid. In certain embodiments, compounds and compositions are administered to the brain parenchyma. In certain embodiments, compounds and compositions are delivered to an animal by intrathecal administration or intracerebroventricular administration. Wide distribution of the compounds and compositions, described herein, within the central nervous system can be achieved by intraparenchymal administration, intrathecal administration or intracerebroventricular administration.
[000248] In certain embodiments, parenteral administration occurs by injection. The injection can be delivered by a syringe or a pump. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly to tissue, such as the striatum, caudate, cortex, hippocampus and cerebellum.
[000249] The mean effective concentration (EC50) of an antisense compound to inhibit huntingtin mRNA expression was calculated after ICV infusion or bolus injection (see Examples 9 and 10). The EC50 for the compound after intrastriatal injection was determined to be 0.45 μg/g. The EC50 after ICV administration was determined to be 26.4 μg/g.
[000250] Therefore, in certain embodiments, the release of a compound or composition described herein may affect the pharmacokinetic profile of the compound or composition. In certain embodiments, injecting a compound or composition described herein into a targeted tissue improves the pharmacokinetic profile of the compound or composition compared to infusing the compound or composition. In a certain embodiment, injection of a compound or composition improves potency compared to wide diffusion, requiring less of the compound or composition to achieve similar pharmacology. In certain embodiments, similar pharmacology refers to the amount of time a target mRNA and/or target protein is down-regulated (e.g., duration of action). In certain embodiments, methods of specifically localizing a pharmaceutical agent, such as by bolus injection, decrease the mean effective concentration (EC50) by a factor of about 50 (e.g., 50 times less tissue concentration is required for achieve the same or similar pharmacodynamic effect). In certain embodiments, methods of specifically localizing a pharmaceutical agent, such as by injection and bolus, decrease the mean effective concentration (EC50) by a factor of 20, 25, 30, 35, 40, 45, or 50. embodiments, the pharmaceutical agent in an antisense compound as further described herein. In certain embodiments, the targeted tissue is brain tissue. In certain embodiments, the targeted tissue is striatal tissue. In certain embodiments, decreasing the EC50 is desirable because it reduces the dose required to achieve a pharmacological outcome in a patient in need thereof.
[000251] The half-life of oligonucleotides and MOE gapmer in brain tissue is 20 days (see Examples 9 to 11). Duration of action as measured by huntingtin mRNA inhibition is prolonged in the brain (see Examples 9 and 10). Intracerebroventricular infusion of antisense oligonucleotides for 2 weeks results in inhibition of huntingtin mRNA by at least 50% in the striatal tissue of BACHD mice for at least 91 days after completion of dosing. Administration by bolus injection resulted in a similar duration of action.
[000252] In certain embodiments, release of a compound or composition, as described herein, into the CNS results in 47% down-regulation of a target mRNA and/or target protein for at least 91 days. In certain embodiments, release of a compound or composition results in at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75% down-regulation of a target mRNA and/or target protein for at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least 100 days, at least 110 days, at least 120 days. In certain embodiments, release to the CNS occurs by intraparenchymal administration, intrathecal administration, or intracerebroventricular administration.
[000253] In certain embodiments, an antisense oligonucleotide is released by injection or infusion once a month, every two months, every 90 days, every 3 months, every 6 months, twice a year or once a year. Certain Combination Therapies
[000254] In certain embodiments, one or more pharmaceutical compositions are co-administered with one or more other pharmaceutical agents. In certain embodiments, one or more other pharmaceutical agents are designed to treat the same disease, disorder or condition as the one or more pharmaceutical compositions written herein. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a disease, disorder or condition other than the one or more pharmaceutical compositions described herein. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an unwanted side effect of one or more pharmaceutical compositions as described herein. In certain embodiments, one or more pharmaceutical compositions are co-administered with another pharmaceutical agent to treat an unwanted effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions are co-administered with another pharmaceutical agent to produce a combinatorial effect. In certain embodiments, one or more pharmaceutical compositions are co-administered with another pharmaceutical agent to produce a synergistic effect.
[000255] In certain embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions and one or more other agents are prepared separately.
[000256] In certain embodiments, pharmaceutical agents that can be co-administered with a pharmaceutical composition that includes antipsychotic agents such as, for example, haloperidol, chlorpromazine, clozapine, quetapine and olanzapine; antidepressant agents such as, for example, fluoxetine, sertraline hydrochloride, venlafaxine and nortriptyline; tranquilizing agents such as, for example, benzodiazepines, clonazepam, paroxetine, venlafaxine and beta-blockers; mood stabilizing agents such as, for example, lithium, valproate, lamotrigine and carbamazepine; paralytic agents such as, for example, Botulinum toxin and/or other experimental agents including, but not limited to, tetrabenazine (Xenazine), creatine, conezyme Q10, trehalose, docosahexanoic acids, ACR16, ethyl-EPA, atomoxetine, citalopram, dimebon , memantine, sodium phenylbutyrate, ramelteon, ursodiol, zyprexa, xenasine, tiapride, riluzole, amantadine, [123I]MNI-420, atomoxetine, tetrabenazine, digoxin, detromethorphan, warfarin, alprozam, ketoconazole, minomepine. EXAMPLES Non-Limiting Disclosure and Incorporation by Reference
[000257] While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references reported in this application is incorporated herein by reference in their entirety. Example 1: Antisense Oligonucleotides Targeting Human Huntingtin Gene Sequences
[000258] About seventeen hundred newly designed antisense compounds of various lengths, motifs and secondary composition targeting human huntingtin gene sequences that have been tested for their effect on human huntingtin mRNA in vitro in various cell types. These gapmers were further designed with internucleoside linkages that are either the phosphorothioate linkages alone (described in Table 1) or that are the phosphorothioate and phosphodiester linkages (described in Table 5). A number of newly designed oligos and two brand name oligonucleotides (previously designed and disclosed) are provided in Tables 1 and 5. Gapmers with fully phosphorothioate internucleoside linkages
Certain of the compounds shown in Table 1 have a motif of 5-10-5 MOE, 6-8-6 MOE, or 5-8-5 MOE. The 5-10'5 gapmers have twenty linked nucleosides, where the central clearance segment has ten 2'-deoxynucleosides and is flanked on both sides (in the 5' and 3' directions) by wings having five nucleosides each. The 6-8-6 gapmer has twenty linked nucleosides, where the central clearance segment has eight 2'-deoxynucleosides and is flanked on both sides (in the 5' and 3' directions) by wings having six nucleosides each. The 5-8-5 gapmers have eighteen linked nucleosides, where the central gap segment has eight 2'-deoxynucleosides and is flanked on both sides (in the 5' and 3' directions) by wings having five nucleosides each. For all gapmers listed in Table 1, each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a 2'-MOE modification. Internucleoside linkages throughout each gapmer are phosphorothioate internucleoside linkages (P=S). All cytokines in each gapmer are 5-methylcytokines. Each gapmer in Table 1 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM_002111.6) or SEQ ID NO: 2 (GENBANK Accession No. NT_006081.17 truncated from nucleotides 462000 to 634000). The 'start site' indicates the 5' most nucleotide where the gapmer is targeted in the human gene sequence No. The 'stop site' indicates the 3' most nucleotide where the gapmer is targeted in the sequence of the human gene N°. Table 1 Chimeric antisense oligonucleotides with phosphorothioate internucleoside linkages targeting human huntingtin gene sequences (SEQ ID NO: 1 and 2)


[000260] The complementarity of gapmers in Table 1 with mouse, rhesus monkey and rat huntingtin gene sequences is further described in Tables 2, 3 and 4.
[000261] The gapmers in Table 2 are complementary with mouse huntingtin mRNA (GENBANK Accession No. NM_010414.1, projected in this as SEQ ID No.: 3). The 'mouse target start site' indicates the 5' most nucleotide where the gapmer is targeted in mouse mRNA. The 'mouse target break site' indicates the 3' most nucleotide where the gapmer is targeted in mouse mRNA. The 'human target start site' indicates the 5' most nucleotide where the gapmer is targeted in the human gene sequence No. The 'human target break site' indicates the 3' most nucleotide where the gapmer is targeted in the human gene sequence No. The 'number of errors' indicates the number of errors between the human oligonucleotide and the mouse mRNA sequence. Table 2 Complementarity of antisense oligonucleotides having phosphorothioate linkages with murine mRNA (SEQ ID NO: 3)


[000262] The gapmers in Table 3 are complementary with the rhesus monkey huntingtin genomic sequence (the complement of GENBANK Accession No. NW_001109716.1 truncated at nucleotides 698000 to 866000, projected in this as SEQ ID No.: 4). The 'Reso Monkey Target Starting Site' indicates the 5' most nucleotide where the gapmer is targeted in the rhesus monkey gene sequence. The 'rhesus monkey target stop site' indicates the 3' most nucleotide where the gapmer is targeted in the rhesus monkey gene sequence. The 'human target start site' indicates the 5' most nucleotide where the gapmer is targeted in the human gene sequence No. The 'human target break site' indicates the 3' most nucleotide where the gapmer is targeted in the human gene sequence No. The 'number of errors' indicates the number of errors between the human oligonucleotide and the rhesus monkey gene sequence. Table 3 Complementarity of antisense oligonucleotides having phosphorothioate linkages with rhesus monkey gene sequence (SEQ ID NO: 4)

[000263] The gapmers in Table 4 are complementary with mouse huntingtin mRNA (GENBANK Accession No. NM_024357.2, projected here as SEQ ID No.: 5). The 'rat target start site' indicates the most 5' nucleotide where the gapmer is targeted in rat mRNA. The 'rat target break site' indicates the 3' most nucleotide where the gapmer is targeted in rat mRNA. The 'human target start site' indicates the 5' most nucleotide where the gapmer is targeted in the human gene sequence No. The 'human target break site' indicates the 3' most nucleotide where the gapmer is targeted in the human gene sequence No. The 'number of errors' indicates the number of errors between the human oligonucleotide and the mouse mRNA sequence. Table 4 Complementarity of antisense oligonucleotides having phosphorothioate linkages with rat mRNA (SEQ ID NO: 5)
Gapmers with mixed phosphorothioate and phosphodiester internucleoside linkages
[000264] The chimeric antisense oligonucleotides in Table 5 were designed as 5-10-5 MOE gapmers. The 5-10-5 gapmers have twenty linked nucleosides, where the central clearance segment has ten 2'-deoxynucleotides and is flanked on both sides (in the 5' and 3' directions) by wings having five nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a 2'-MOE modification. The internucleoside bonds within the central overhang segment, the bonds connected to the 5' overhang segment or 3' wing segment, and the bonds across the 5' plus 3' nucleosides of each wing segment are all phosphorothioate bonds ( P=S); the internucleoside bonds connecting the remainder of the nucleosides from both the 5' and 3' wing segments are phosphodiester bonds; ie the gapmer has a mixed backbone. All cytokines in each gapmer are 5-methylcytokines. Each gapmer in Table 5 is targeted to human mRNA sequence (GENBANK Accession No. NM_002111.6, projected herein as SEQ ID No.: 1). The 'start site' indicates the most 5' nucleotide where the gapmer is targeted in human mRNA#. The 'stop site' indicates the 3' most nucleotide where the gapmer is targeted in human mRNA#. Table 5 Chimeric antisense oligonucleotides with phosphorothioate and internucleoside phosphate linkages targeting human huntingtin mRNA (SEQ ID NO: 1)

[000265] The complementarity of gapmers in Table 5 with mouse, rhesus monkey and rat huntingtin gene sequences are further described in Tables 6, 7 and 8.
[000266] The gapmers in Table 6 are complementary with mouse huntingtin mRNA (GENBANK Accession No. NM_010414.1; SEQ ID No.: 3). The 'mouse target start site' indicates the 5' most nucleotide where the gapmer is targeted in mouse mRNA. The 'mouse target break site' indicates the 3' most nucleotide where the gapmer is targeted in mouse mRNA. The 'human target start site' indicates the 5' most nucleotide where the gapmer is targeted in human mRNA (GENBANK Accession No. NM_002111.6). The 'human target break site' indicates the 3' most nucleotide where the gapmer is targeted in human mRNA (GENBANK Accession No. NM_002111.6). The 'number of errors' indicates the number of errors between the human oligonucleotide and the mouse mRNA sequence. Table 6 Complementarity of antisense oligonucleotides having mixed phosphorothioate and phosphate linkages with murine mRNA (SEQ ID NO: 3)

[000267] Tabe a 7 gapmers are complementary with the rhesus monkey huntingtin genomic sequence (the complement of GENBANK Accession No. NW_001109716.1 truncated at nucleotides 698000 to 866000; SEQ ID NO: 4). The 'rhesus monkey target start site' indicates the 5' most nucleotide where the gapmer is targeted in the rhesus monkey gene sequence. The 'rhesus monkey target stop site' indicates the 3' most nucleotide where the gapmer is targeted in the rhesus monkey gene sequence. The 'human target start site' indicates the 5' most nucleotide where the gapmer is targeted in human mRNA (GENBANK Accession No. NM_002111.6). The 'human target break site' indicates the 3' most nucleotide where the gapmer is targeted in human mRNA (GENBANK Accession No. NM_002111.6). The 'number of errors' indicates the number of errors between the human oligonucleotide and the rhesus monkey gene sequence. Table 7 Complementarity of antisense oligonucleotides having mixed phosphorothioate and phosphate linkages with rhesus monkey gene sequence (SEQ ID NO: 4)

[000268] The gapmers in Table 8 are complementary with mouse huntingtin mRNA (GENBANK Accession No. NM_024357.2; SEQ ID No.: 5). The 'rat target start site' indicates the most 5' nucleotide where the gapmer is targeted in rat mRNA. The 'rat target break site' indicates the 3' most nucleotide where the gapmer is targeted in rat mRNA. The 'human target start site' indicates the 5' most nucleotide where the gapmer is targeted in human mRNA (GENBANK Accession No. NM_002111.6). The 'human target break site' indicates the 3' most nucleotide where the gapmer is targeted in human mRNA (GENBANK Accession No. NM_002111.6). The 'number of errors' indicates the number of errors between the human oligonucleotide and the mouse mRNA sequence. Table 8
Example 2: Dose-dependent antisense inhibition of human huntingtin NA mR in vitro
[000269] Approximately one thousand seven hundred newly designed antisense compounds of various lengths, motifs and secondary composition have been tested for their effect on human huntingtin mRNA in vitro in various cell types. These compounds were compared to about two hundred and fifty previously designed compounds including the compound ISIS 387916 which was previously determined to be a compound of considerable potency in vivo. As shown in this example, ISIS 419640, ISIS 419641, ISIS 419642, ISIS 436665, ISIS 436671, ISIS 436689, ISIS 437507, ISIS 443139, ISIS 444591, ISIS 444661, ISIS 437527, ISIS 444584 and ISIS 444652 and previously designed ISIS 388241 were observed have better or similar potency than the ISIS 387916 reference mark compound in vitro. Fibroblasts GM04281
[000270] GM04281 fibroblasts cultured at a density of 25,000 cells per well were transfected using electroporation with 500 nM, 1000 nM, 2000 nM, 4000 nM, or 8000 nM of the antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA levels were measured by quantitative real-time PCR. The human primer probe series RTS2617 (advanced sequence CTCCGTCCGGTAGACATGCT, designed herein as SEQ ID NO: 37; reverse sequence GGAAATCAGAACCCTCAAAATGG, designed herein as SEQ ID NO: 38; probe sequence TGAGCACTGTTCAACTGTGGATATCGGGAX, design herein as SEQ ID NO: 39 ) was used to measure mRNA levels. Huntingtin mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. The results are shown in Table 9 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction of huntingtin mRNA levels.
[000271] The half maximum inhibitory concentration (IC50) of each oligonucleotide is also shown in Table 9 and was calculated by plotting the concentrations of the oligonucleotides used versus the percentage inhibition of huntingtin mRNA expression achieved at each concentration and observing the concentration of oligonucleotides at which 50% inhibition of huntingtin mRNA expression was achieved compared to control. The IC50 is expressed in µM. Table 9 Dose-dependent reduction of huntingtin mRNA in GM04281 Fibroblasts

[000272] ISIS 387916, ISIS 388241 and ISIS 437507 were further tested for their effect on human huntingtin mRNA in vitro. Cultured GM04281 fibroblasts were tested in a similar procedure as described above. The results are shown in Table 10 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction of huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 10 expressed in µM. Table 10 Dose-dependent reduction of huntingtin mRNA in GM04281 Fibroblasts

[000273] ISIS 387916, ISIS 388241 and ISIS 437507 were further tested for their effect on human huntingtin mRNA in vitro. Cultured GM04281 fibroblasts were tested in a similar procedure as described above. Results are shown in Table 11 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction of huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 11 expressed in µM. Table 11 Dose-dependent reduction of huntingtin mRNA in GM04281 Fibroblasts

[000274] ISIS 387916, ISIS 388241, ISIS 419641 and ISIS 436754 were further tested for their effect on human huntingtin mRNA in vitro. Cultured GM04281 fibroblasts were tested in a similar procedure as described above. The results are shown in Table 12 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction in huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 12 expressed in µM. Table 12 Dose-dependent reduction of huntingtin mRNA in GM04281 Fibroblasts

[000275] ISIS 387916, ISIS 388241 and ISIS 437507 were further tested for their effect on human huntingtin mRNA in vitro. GM04281 fibroblasts cultured at a density of 25,000 cells per well were transfected using electroporation with 250 nM, 500 nM, 1000 nM, 2000 nM, 4000 nM or 8000 nM of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA levels were measured by quantitative real-time PCR. The RTS2617 human primer probe series was used to measure mRNA levels. Huntingtin mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. The results are shown in Table 13 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction of huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 13 expressed in µM. Table 13 Dose-dependent reduction of huntingtin mRNA in GM04281 Fibroblasts

[000276] ISIS 387916, ISIS 388241, ISIS 419628, ISIS 419629, ISIS 419637, ISIS 436684, ISIS 443139, ISIS 444584, ISIS 444615, ISIS 444627, ISIS 444652, ISIS 444658, ISIS 444659, ISIS 444660 and ISIS 444661 were still tested for its effect on human huntingtin mRNA in vitro. GM04281 fibroblasts cultured at a density of 25,000 cells per well were transfected using electroporation with 156.25 nM, 312.5 nM, 625 nM, 1250 nM, or 2500 nM of the antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA levels were measured by quantitative real-time PCR. The RTS2617 human primer probe series was used to measure mRNA levels. Huntingtin mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. The results are shown in Table 14 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction in huntingtin mRNA levels. The data presented is the average of two experiments. The IC50 of each antisense oligonucleotide is also shown in Table 14 expressed in µM. Table 14 Dose-dependent reduction of huntingtin mRNA in GM04281 Fibroblasts

[000277] ISIS 387916, ISIS 436671, ISIS 444661, SIS 41964 and ISIS 436665 were further tested for their effect on human huntingtin mRNA in vitro. GM04281 fibroblasts cultured at a density of 25,000 cells per well were transfected using electroporation with 13,6719 nM, 27,3438 nM, 54,6875 nM, 109,375 nM, 218.75 nM, 437.5 nM, 875 nM, 1750 nM, 3500 nM, or 7000 nM of the oligonucleotide anti-sense. After a treatment period of approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA levels were measured by quantitative real-time PCR. The RTS2617 human primer probe series was used to measure mRNA levels. Huntingtin mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. The results are shown in Table 15 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction of huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 15 expressed in µM. Table 15 Dose-dependent reduction of huntingtin mRNA in GM04281 Fibroblasts

[000278] ISIS 387916, ISIS 388241, ISIS 437168 and ISIS 437175 were further tested for their effect on human huntingtin mRNA in vitro. GM04281 fibroblasts cultured at a density of 25,000 cells per well were transfected using electroporation with 250 nM, 500 nM, 1000 nM, 2000 nM, 4000 nM and 8000 nM of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA levels were measured by quantitative real-time PCR. The RTS2617 human primer probe series was used to measure mRNA levels. Huntingtin mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. The results are presented in Table 15.1 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction of huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 15.1 expressed in µM. Table 15.1 Dose-dependent reduction of huntingtin mRNA in GM04281 Fibroblasts

[000279] ISIS 387916, ISIS 388241, ISIS 437441 and ISIS 437442 were further tested for their effect on human huntingtin mRNA in vitro. Cultured GM04281 fibroblasts were tested in a similar procedure as described above. Results are presented in Table 15.2 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction of huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 15.2 expressed in µM. Table 15.2 Dose-dependent reduction of huntingtin mRNA in GM04281 Fibroblasts

[000280] ISIS 387916, ISIS 388241, ISIS 437175 and ISIS 437527 were further tested for their effect on human huntingtin mRNA in vitro. Cultured GM04281 fibroblasts were tested in a similar procedure as described above. The results are shown in Table 15.3 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction of huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 15.3 expressed in µM. Table 15.3 Dose-dependent reduction of huntingtin mRNA in GM04281 Fibroblasts

[000281] Some of the antisense oligonucleotides described in Example 1 were tested for their effect on human huntingtin mRNA in vitro. A549 cells grown at a density of 4,000 cells per well were transfected using lipofectin transfection reagent with 7,4074 nM, 22,222 nM, 66,667 nM, or 200 nM of the antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA levels were measured by quantitative real-time PCR. The RTS2617 human primer probe series was used to measure mRNA levels. Huntingtin mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. The results are shown in Table 16 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction in huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 16 expressed in nM. Table 16 Dose-dependent reduction of huntingtin mRNA in A549 cells

[000282] ISIS 387916, ISIS 388241 and ISIS 437507 were further tested for their effect on human huntingtin mRNA in vitro. A549 cells grown at a density of 20,000 cells per well were transfected using electroporation with 250 nM, 500 nM, 1000 nM, 2000 nM, 4000 nM or 8000 nM of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA levels were measured by quantitative real-time PCR. The RTS2617 human primer probe series was used to measure mRNA levels. Huntingtin mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are shown in Table 17 expressed as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction in huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 17 expressed in µM. Table 17
LLC-MK2 Cells
[000283] Some of the antisense oligonucleotides described in Example 1 and targeted to human huntingtin nucleic acid were tested for their effect on huntingtin mRNA rhesus in vitro. LLC-MK2 cells grown at a density of 25,000 cells per well were transfected using electroporation with 625 nM, 1250 nM, 2500 nM, 5000 nM, 10,000 nM, or 20,000 nM of the antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA levels were measured by quantitative real-time PCR. The human primer probe series RTS2686 (advanced sequence GTCTGAGCCTCTCTCGGTCAA, designed herein as SEQ ID NO: 40; reverse sequence AAGGGATGCTGGGCTCTGT, designed herein as SEQ ID NO: 41; probe sequence AGCAAAGCTTGGTGTCTTGGCACTGTTAGTX, design herein as SEQ ID NO: 42 ) was used to measure mRNA levels. Huntingtin mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. The results are shown in Table 18 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction in huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 18 expressed in µM. Table 18

[000284] ISIS 3 87916, ISIS 388241, ISIS 436684, SIS 437168, ISIS 437175, ISIS 437441, ISIS 437507, ISIS 437527, ISIS 444578, ISIS 444584, ISIS 444591 and ISIS 444607 were further tested for their effect on reso mRNA huntingtin in vitro. Cultured LLC-MK2 cells were tested in a similar procedure as described above. The results are shown in Table 19 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction of huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 19 expressed in µM. Table 19
nd= IC50 should not be measured by that compound
[000285] ISIS 387916, ISIS 388241, ISIS 444608, ISIS 444615, ISIS 444618, ISIS 444627, ISIS 444652, ISIS 444658, ISIS 444659, ISIS 444660 and ISIS 444661 were further tested for their effect on in vitro reso-monkey huntingtin mRNA. Cultured LLC-MK2 cells were tested in a similar procedure as described above. The results are shown in Table 20 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction in huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 20 expressed in µM. Table 20 Dose-dependent reduction of huntingtin mRNA in LLC-MK2 cells

[000286] ISIS 387916, ISIS 419627, ISIS 419628, ISIS 419629, ISIS 419630, ISIS 419636, ISIS 419637, ISIS 419640, ISIS 419641 and ISIS 419642 were further tested for their effect on reso monkey huntingtin mRNA in vitro. LLC-MK2 cells grown at a density of 3000 cells per well were transfected using lipofectin transfection reagent with 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, or 200 nM of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA levels were measured by quantitative real-time PCR. The RTS2686 human primer probe series was used to measure mRNA levels. Huntingtin mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. The results are shown in Table 21 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction in huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 21 expressed in nM. Table 21

[000287] ISIS 387916, ISIS 419641 and ISIS 436689 were further tested for their effect on rhesus monkey huntingtin mRNA in vitro. LLC-MK2 cells grown at a density of 3000 cells per well were transfected using LipofectAMINE2000 transfection reagent with 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, or 200 nM of the antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA levels were measured by quantitative real-time PCR. The RTS2686 human primer probe series was used to measure mRNA levels. Huntingtin mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. The results are shown in Table 22 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction of huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 22 expressed in nM. Table 22

[000288] ISIS 387916, ISIS 388241, ISIS 436665, ISIS 436671 and ISIS 436689 were further tested for their effect on rhesus monkey huntingtin mRNA in vitro. LLC-MK2 cells grown at a density of 3,000 cells per well were transfected using lipofectin transfection reagent with either 46875 nM, 9,375 nM, 18.75 nM, 37.5 nM, 75 nM, or 150 nM of the antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA levels were measured by quantitative real-time PCR. The RTS2686 human primer probe series was used to measure mRNA levels. Huntingtin mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. The results are shown in Table 23 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction in huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 23 expressed in nM. Table 23 Dose-dependent reduction of huntingtin mRNA in LLC-MK2 cells
BACHD transgenic mouse hepatocytes
[000289] Some of the antisense oligonucleotides described in Example 1 and targeted to human huntingtin nucleic acid were tested for their effect on human huntingtin mRNA in vitro. BACHD mouse hepatocytes cultured at a density of 10,000 cells per well were transfected using cytofectin transfection reagent 7,4074 nM, 22,222 nM, 66,667 nM, or 200 nM of the antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA levels were measured by quantitative real-time PCR. The RTS2617 human primer probe series was used to measure mRNA levels. Huntingtin mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are shown in Table 24 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction of huntingtin mRNA levels. The data presented is the average of two experiments. The IC50 of each antisense oligonucleotide is also shown in Table 24 expressed in nM. Table 24 Dose-dependent reduction of huntingtin mRNA in BACHD transgenic murine hepatocytes

[000290] ISIS 387916, ISIS 388241 and ISIS 419641 were further tested for their effect on human huntingtin mRNA in vitro. BACHD mouse hepatocytes cultured at a density of 10,000 cells per well were transfected using 12.5 nM, 25 nM, 50 nM, 100 nM or 200 nM cytofectin transfection reagent of the antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA levels were measured by quantitative real-time PCR. The RTS2617 human primer probe series was used to measure mRNA levels. Huntingtin mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. The results are shown in Table 25 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction in huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 25 expressed in nM. Table 25 Dose-dependent reduction of huntingtin mRNA in BACHD transgenic murine hepatocytes

[000291] ISIS 38791 6, ISIS 388241, ISIS 41964 H, ISIS 436665, ISIS 436671 and ISIS 436689 were further tested for their effect on human huntingtin mRNA in vitro. Cultured BACHD mouse hepatocytes were tested in an identical manner as described above. The results are shown in Table 26 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction in huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 26 expressed in nM. Table 26 Dose-dependent reduction of huntingtin mRNA in BACHD transgenic murine hepatocytes

[000292] ISIS 387916, ISIS 419640, ISIS 419641 and ISIS 419642 were further tested for their effect on mouse huntingtin mRNA in vitro. BACHD mouse hepatocytes cultured at a density of 20,000 cells per well were transfected using 6667 nM, 20 nM, 60 nM, or 180 nM cytofectin transfection reagent of the antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA levels were measured by quantitative real-time PCR. The murine primer probe series RTS2633 (advanced sequence CAGAGCTGGTCAACCGTATCC, designed herein as SEQ ID NO: 43; reverse sequence GGCTTAAACAGGGAGCCAAAA, designed herein as SEQ ID NO: 44; probe sequence ACTTCATGATGAGCTCGGAGTTCAACX, designed herein as SEQ ID NO:: 44; 45) was used to measure mRNA levels. Huntingtin mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are shown in Table 27 as percentage inhibition of huntingtin mRNA relative to untreated control cells and demonstrate antisense oligonucleotide-mediated dose-dependent reduction of huntingtin mRNA levels. The IC50 of each antisense oligonucleotide is also shown in Table 27 expressed in nM. Table 27 Dose-dependent reduction of huntingtin mRNA in BACHD transgenic murine hepatocytes
Example 3: Systemic administration of antisense oligonucleotides against huntingtin mRNA in BACHD mice
[000293] About one thousand seven hundred newly designed antisense compounds, sixty-six compounds were selected based on in vitro potency compared to ISIS 387916 for testing in systemic tolerability assessments.
[000294] BACHD mice were treated with ISIS oligonucleotides and evaluated for changes in the levels of various metabolic markers as well as inhibition of huntingtin mRNA in the liver. Antisense oligonucleotides that cause adverse changes in body weight, organ weight, or levels of metabolic markers were considered unsuitable for use in the additional studies. Study 1.Treatment
[000295] Nineteen groups of four BACHD mice each were injected intraperitoneally with 12.5 mg/kg of ISIS 387916, ISIS 388241, ISIS 419629, ISIS 419637, ISIS 436684, ISIS 444578, ISIS 444584, ISIS 444591, ISIS 444607, ISIS 444608 , ISIS 444615, ISIS 444618, ISIS 444627, ISIS 444652, ISIS 444658, ISIS 444659, ISIS 444660, ISIS 444661, or ISIS 444663 twice a week for 2 weeks. A control group of four mice was injected intraperitoneally with PBS twice a week for 2 weeks. Two days after the last dose, the mice were anesthetized with isoflurane and exsanguinated by plasma collection, after which cervical dislocation was performed and the organs collected. RNA analysis
[000296] RNA was extracted from liver tissue for real-time PCR analysis of huntingtin mRNA levels. Human mutant huntingtin mRNA levels were measured using a series of human RTS2617 primers. Mouse normal huntingtin levels were measured using the RTS2633 mouse primer probe series. The results are shown in Tables 28 and 29 and were calculated as percentage human inhibition and murine huntingtin expression levels, respectively, relative to the PBS control. All antisense oligonucleotides effect significant inhibition of human huntingtin mRNA levels. ISIS 388241 has more than three errors with murine huntingtin mRNA (SEQ ID NO: 3) and therefore does not show significant inhibition of murine mRNA levels compared to control. Table 28 Percent inhibition of human huntingtin mRNA in BACHD mice
Table 29 Percent inhibition of murine huntingtin mRNA in BACHD mice

Organ Weight Measurements
[000297] Kidney, spleen, liver weights were measured at the end of the study and are presented in Table 30 as a percentage of saline control normalized to body weight. Table 30 Percentage change in organ weight of BACHD mice after antisense oligonucleotide treatment
Liver Function Assessment
[000298] To assess the impact of ISIS oligonucleotides on liver function in the mice described above, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). Alanine transaminase (ALT) and aspartate transaminase (AST) measurements are expressed in IU/L and the results are shown in Table 31. Table 31 Effect of antisense oligonucleotide treatment on liver function markers
Study 2 Treatment
[000299] Fourteen groups of four BACHD mice each were injected intraperitoneally with 12.5 mg/kg or 50 mg/kg of ISIS 419581, ISIS 419602, ISIS 419628, ISIS 419629, ISIS 419640, ISIS 419641, or ISIS 419642 twice in week for 2 weeks. A group of four BACHD mice were injected intraperitoneally with 12.5 mg/kg of ISIS 387916 twice a week for 2 weeks. A control group of four mice was injected intraperitoneally with PBS twice a week for 2 weeks. Two days after the last dose, the mice were anesthetized with isoflurane and exsanguinated by plasma collection, after which cervical dislocation was performed and the organs collected. RNA analysis
[000300] RNA was extracted from liver tissue for real-time PCR analysis of huntingtin mRNA levels. Human mutant huntingtin mRNA levels were measured using a series of human RTS2617 primers. Mouse normal huntingtin levels were measured using the RTS2633 mouse primer probe series. The results are shown in Tables 32 and 33 and were calculated as percentage human inhibition and murine huntingtin expression levels, respectively, relative to PBS control. Table 32 Percent Inhibition of Human Huntingtin mRNA in BACHD mice
Table 33 Percentage inhibition of murine huntingtin mRNA in BACHD mice

[000301] Kidney, spleen, liver weights were measured at the end of the study and are presented in Table 34 as a percentage of saline control normalized to body weight. Table 34 Percentage change in organ weight of BACHD mice after antisense oligonucleotide treatment


[000302] To assess the impact of ISIS oligonucleotides on liver function in the mice described above, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). ALT and AST measurements are expressed in IU/L and the results are shown in Table 35. Table 35
Study 3 Treatment
[000303] Eighteen groups of four BACHD mice each were injected intraperitoneally with 12.5 mg/kg or 50 mg/kg of ISIS 388250, ISIS 388251, ISIS 388263, ISIS 388264, ISIS 419641, ISIS 436645, ISIS 436649, ISIS 436668, or ISIS 436689 twice a week for 2 weeks. A group of four BACHD mice were injected intraperitoneally with 12.5 mg/kg of ISIS 388241 twice a week for 2 weeks. A control group of four mice was injected intraperitoneally with PBS twice a week for 2 weeks. Two days after the last dose, the mice were anesthetized with isoflurane and exsanguinated by plasma collection, after which cervical dislocation was performed and the organs collected. RNA analysis
[000304] RNA was extracted from liver tissue for real-time PCR analysis of huntingtin mRNA levels. Human mutant huntingtin mRNA levels were measured using a series of human RTS2617 primers. Mouse normal huntingtin levels were measured using the RTS2633 mouse primer probe series. The results are shown in Tables 36 and 37 and were calculated as percentage human inhibition and murine huntingtin expression levels, respectively, relative to PBS control. All antisense oligonucleotides effect significant inhibition of human huntingtin mRNA levels. ISIS 388241, ISIS 388250, ISIS 388251, ISIS 388263, ISIS 388264 and ISIS 436645 have more than three errors with murine huntingtin mRNA (SEQ ID NO: 3) and therefore show no significant inhibition of murine mRNA levels compared to the control. ISIS 436649 and ISIS 436689 have three errors with murine huntingtin mRNA (SEQ ID NO: 3) and therefore do not show significant inhibition of murine mRNA levels compared to control. Table 36 Percentage inhibition of human huntingtin mRNA in BACHD mice
Table 37 Percent inhibition of murine huntingtin mRNA in BACHD mice

[000305] Kidney, spleen, liver weights were measured at the end of the study and are presented in Table 38 as a percentage of saline control normalized to body weight. The mice treated with ISIS 388263 and ISIS 436645 had an increase in liver weight at a dosage of 50 mg/kg compared to the PBS control. Table 38 Percentage change in organ weight of BACHD mice after antisense oligonucleotide treatment
Liver Function Assessment
Estudo 4 T ratamento[000306] To assess the impact of ISIS oligonucleotides on liver function in the mice described above, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). Alanine transaminase (ALT) and aspartate transaminase (AST) measurements are expressed in IU/L and the results are shown in Table 39. Table 39 Effect of antisense oligonucleotide treatment on liver function markers
Study 4 Treatment
[000307] Eighteen groups of four BACHD mice each were injected intraperitoneally with 12.5 mg/kg or 50 mg/kg of ISIS 388241, ISIS 437123, ISIS 437132, ISIS 437140, ISIS 437442, ISIS 437446, ISIS 437477, ISIS 437478, or ISIS 437490 twice a week for 2 weeks. A group of four BACHD mice were injected intraperitoneally with 12.5 mg/kg of ISIS 387916 twice a week for 2 weeks. A control group of four mice was injected intraperitoneally with PBS twice a week for 2 weeks. Two days after the last dose, the mice were anesthetized with isoflurane and exsanguinated by plasma collection, after which cervical dislocation was performed and the organs collected. RNA analysis

Tabela 41 Percentual de inibição de mRNA de huntingtina de murino em camundongos BACHD
[000308] RNA was extracted from liver tissue for real-time PCR analysis of huntingtin mRNA levels. Human mutant huntingtin mRNA levels were measured using a series of human RTS2617 primers. Mouse normal huntingtin levels were measured using the RTS2633 mouse primer probe series. The results are shown in Tables 40 and 41 and were calculated as percentage human inhibition and murine huntingtin expression levels, respectively, relative to the PBS control. ISIS 388241 and ISIS 437490 have more than three errors with murine huntingtin mRNA (SEQ ID NO: 3) and therefore do not show significant inhibition of murine mRNA levels compared to control. ISIS 437132 has three errors with murine huntingtin mRNA (SEQ ID NO: 3) and therefore does not show significant inhibition of murine mRNA levels compared to control. ISIS 437123 and ISIS 437140 have two errors with murine huntingtin mRNA (SEQ ID NO: 3) and do not show significant inhibition of murine mRNA levels compared to control. Table 40
Table 41 Percentage inhibition of murine huntingtin mRNA in BACHD mice
Organ Weight Measurements
[000309] Kidney, spleen, liver weights were measured at the end of the study and are presented in Table 42 as a percentage of saline control normalized to body weight. Table 42 Percentage change in organ weight of BACHD mice after antisense oligonucleotide treatment

Liver Function Assessment
Estudo 5 T ratamento[000310] To assess the impact of ISIS oligonucleotides on liver function in the mice described above, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). Alanine transaminase (ALT) and aspartate transaminase (AST) measurements are expressed in IU/L and the results are shown in Table 43. Table 43 Effect of antisense oligonucleotide treatment on liver function markers Study 5 Treatment
[000311] Eleven groups of four BACHD mice each were injected intraperitoneally with 12.5 mg/kg of ISIS 388241, ISIS 419640, ISIS 419641, ISIS 419642, ISIS 436665, ISIS 436671, ISIS 436689, ISIS 437507, ISIS 443139, ISIS 444591 , or ISIS 444661 twice a week for 2 weeks. A control group of four mice was injected intraperitoneally with phosphate buffered saline (PBS) twice a week for 2 weeks. Two days after the last dose, the mice were anesthetized with isoflurane and exsanguinated by plasma collection, after which cervical dislocation was performed and the organs collected. RNA analysis
Tabela 45 Percentual de inibição de mRNA de huntingtina de murino em camundongos BACHD [000312] RNA was extracted from liver tissue for real-time PCR analysis of huntingtin mRNA levels. Human mutant huntingtin mRNA levels were measured using a series of human RTS2617 primers. Mouse normal huntingtin levels were measured using the RTS2633 mouse primer probe series. The results are presented in Tables 44 and 45 and were calculated as percentage human inhibition and murine huntingtin expression levels, respectively, relative to PBS control. All antisense oligonucleotides effect significant inhibition of human huntingtin mRNA levels. ISIS 388241, ISIS 437507 and ISIS 443139 have more than three errors with murine huntingtin mRNA (SEQ ID NO: 3) and therefore do not show significant inhibition of murine mRNA levels compared to control. ISIS 436689 has 3 errors with murine huntingtin mRNA (SEQ ID NO: 3) and does not show significant inhibition of murine mRNA levels compared to control. Table 44 Percentage of inhibition of human huntingtin mRNA in BACHD mice Table 45 Percentage inhibition of murine huntingtin mRNA in BACHD mice
[000313] Body weight and organ weight measurements
[000314] The body weights of mice were measured at baseline and twice weekly thereafter. The mouse body weights are shown in Table 46 and are expressed as a percentage change in weights taken at baseline. The results indicate that treatment with these oligonucleotides did not cause any adverse reaction in the body weight of mice throughout the study. Table 46 Percentage change in body weight of BACHD mice after antisense oligonucleotide treatment
Avaliação da função do fígado[000315] Kidney, spleen, liver weights were measured at the end of the study and are presented in Table 47 as a percentage of saline control normalized to body weight. Table 47 Percentage change in organ weight of BACHD mice after antisense oligonucleotide treatment Liver Function Assessment
Medição da função hepática[000316] To assess the impact of ISIS oligonucleotides on liver function in the mice described above, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). ALT and AST measurements are expressed in IU/L. The plasma levels of bilirubin and albumin were also measured using the same clinical chemistry analyzer and expressed in g/dL. The results are shown in Table 48. Table 48 Effect of antisense oligonucleotide treatment on liver function markers Liver function measurement
Medição de outros parâmetros metabólicos[000317] To assess the impact of ISIS Oligonucleotides on liver function of mice described above, plasma concentrations of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The results are shown in Table 49 expressed in mg/dL. Table 49 Effect of antisense oligonucleotide treatment on liver function markers BUN Creatinine Measurement of other metabolic parameters
Exemplo 4: Administração de bolo de oligonucleotídeos anti-sentido contra mRNA de huntingtina ao estriado dos camundongos BACHD[000318] To assess the impact of ISIS oligonucleotides on other metabolic functions in mice described above, plasma concentrations of glucose, cholesterol and triglycerides were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The results are shown in Table 50 expressed in mg/dL and demonstrate that treatment with these oligonucleotides does not cause any adverse reactions at the level of these metabolic markers between the control and treatment groups. Table 50 Effect of antisense oligonucleotide treatment on metabolic markers Example 4: Administration of antisense oligonucleotides bolus against huntingtin mRNA to the striatum of BACHD mice
[000319] BACHD mice were treated with ISIS oligonucleotides by administering bolus to a defined mouse brain area, the striatum, for the purpose of evaluating the activity of oligonucleotides in brain tissue against human and mouse huntingtin expressing mRNA. treatment and surgery
[000320] Groups of four BACHD mice each were administered with ISIS 388241, ISIS 419628, ISIS 419637, ISIS 419640, ISIS 419641, ISIS 419642, ISIS 436665, ISIS 436671, ISIS 436684, ISIS 436689, ISIS 436754, ISIS 437168, ISIS 437 , ISIS 437441, ISIS 437442, ISIS 437507, ISIS 437527, ISIS 443139, ISIS 444578, ISIS 444584, ISIS 444591, ISIS 444607, ISIS 444608, ISIS 444608, ISIS 444618, ISIS 444627, ISIS 444652, ISIS 444658, ISIS 444659 444660, ISIS 444661 or ISIS 444663 delivered as a cake injection at 3 μg, 10 μg or 25 μg concentrations in the striatum.
[000321] A control group of 4 BACHD mice were similarly treated with PBS. ISIS 388241 was administered in seven groups of 4 mice each and the results presented are the average of data derived from 28 mice. ISIS 419628 was administered in 2 groups of 4 BACHD mice each and the results presented are the average of data derived from 8 mice. Seven days after bolus administration, mice were euthanized using isoflurane and the organs were removed. The animals were decapitated and the brain was removed for striatal tissue dissection. RNA analysis
[000322] RNA was extracted from striatal tissue for real-time PCR analysis of huntingtin mRNA levels. Human mutant huntingtin mRNA levels were measured using a series of human RTS2617 primers. Normal mouse huntingtin mRNA levels were measured using a series of murine primer probe RTS2633. Results for human huntingtin mRNA levels are shown in Table 51 and are expressed as percent inhibition compared to the PBS control group. All antisense oligonucleotides effect dose-dependent inhibition of human huntingtin mRNA levels. Results for murine huntingtin mRNA levels are shown in Table 52 and are expressed as percent inhibition compared to the PBS control group.
[000323] The effective dosages (ED50) of each oligonucleotide for human huntingtin mRNA and mouse huntingtin mRNA were calculated by plotting the concentrations of ''oligonucleotides used versus the percentage inhibition of expression of species and huntingtin mRNA levels of species and observing the concentrations at which 50% inhibition of huntingtin mRNA expression was achieved for each species compared to the corresponding controls. The ED50 (μg) for each antisense oligonucleotide is also shown in Tables 51 and 52 for murine and human huntingtin mRNA respectively.
Tabela 52 Percentual de inibição dos níveis de mRNA de huntingtina de murino em vivo e ED50 dos oligonucleotídeos anti-sentido [000324] ISIS 388241, ISIS 436684, ISIS 436754, ISIS 437175, ISIS 437507, ISIS 443139 and ISIS 444584 are each mismatched by 8 base pairs or more with murine huntingtin mRNA (SEQ ID NO: 3) and therefore, they do not show significant inhibition of murine mRNA levels compared to control. ISIS 437168 and ISIS 437441 have 2 errors each with murine huntingtin mRNA (SEQ ID NO: 3) and do not show significant inhibition of murine mRNA levels compared to control. ISIS 436689 has 3 errors with murine huntingtin mRNA (SEQ ID NO: 3) and does not show significant inhibition of murine mRNA levels compared to control. Table 51 Percent inhibition of human huntingtin mRNA levels in vivo and ED50 of antisense oligonucleotides Table 52 Percent inhibition of murine huntingtin mRNA levels in vivo and ED50 of antisense oligonucleotides
[000325] The ten compounds marked with an asterisk have an improved ED50 in ISIS 388241.
[000326] Example 5: Assay for neurotoxic effects of bolus administration of antisense oligonucleotides in striatal tissue of rats
[000327] About 30 compounds were selected as having high tolerability and high potency. The compounds were then tested by injecting the CNS bolus into the rat further to assess neurotoxicity.
[000328] Sprague-Dawley rats were each treated with ISIS oligonucleotides by administering bolus to a defined brain area, the striatum, for the purpose of evaluation for the introduction of the microglial marker AIF1 as a measure of CNS toxicity. treatment and surgery
Groups of four Sprague-Dawley rats were administered with ISIS 387916, ISIS 388241, ISIS 419627, ISIS 419628, ISIS 419629, ISIS 419630, ISIS 419636, ISIS 419637, ISIS 419640, ISIS 419641, ISIS 419642, ISIS 436665, ISIS 436668, ISIS 4196671, ISIS 436684, ISIS 436689, ISIS 436754, ISIS 443168, ISIS 437175, ISIS 437441, ISIS 437442, ISIS 437507, ISIS 437507, ISIS 437527, ISIS 443139, ISIS 444578, ISIS 444584, ISIS 464591, ISIS 444607, ISIS 444607 , ISIS 444615, ISIS 444618, ISIS 444627, ISIS 444652, ISIS 444658, ISIS 444659, ISIS 444660, ISIS 444661, or ISIS 444663 released as a cake injection at a concentration of 50 µg in the striatum.
[000330] A control group of 4 mice were similarly treated with PBS. A group of 4 mice were similarly treated with ISIS 104838, an antisense oligonucleotide against TNF-α, as a negative control group. ISIS 387916 was administered into four groups of 4 mice each and the results shown are an average of data derived from 16 mice. ISIS 419628 was administered to two groups of 4 mice each and the results shown are the average of the data from 8 mice. ISIS 419629, ISIS 444584, and ISIS 444618, which have toxic indicators in the systemic administration study (Example 3) were also tested in this study. Seven days after bolus administration, rats were euthanized using isoflurane and organs were removed. The animals were decapitated and the brain was removed for striatal tissue dissection. RNA analysis of AIF1 expression levels
Análise de RNA de níveis de expressão de huntingtina[000331] RNA was extracted from striatal tissue for real-time PCR analysis of AIF1 mRNA levels. Rat AIF1 levels were measured using the rat primer probe series rAif1_LTS00219 (advanced sequence AGGAGAAAAACAAAGAACACCAGAA, projected herein as SEQ ID NO: 46; reverse sequence CAATTAGGGCAACTCAGAAATAGCT, designed herein as SEQ ID NO: 47; probe sequence CCAACTCAGGTCCCCC projected on this as SEQ ID NO:48). The results were calculated as the percentage of AIF1 expression in that of the PBS control and are shown in Table 53. ISIS 419629, ISIS 444584 and ISIS 444618, which have toxic indicators in the systemic administration study (in Example 3), also have toxic indicators in this study (greater than 300% saline control above). Later studies show that ISIS 444584 is neurotolerable and exhibits negligible toxic indicators (see Examples 16 and 17). Table 53 Percent expression of AIF1 mRNA levels in vivo as a measure of neurotoxicity RNA analysis of huntingtin expression levels
[000332] RNA was extracted from striatal tissue for real-time PCR analysis of huntingtin mRNA levels. Rat huntingtin mRNA levels were measured using the rat primer probe series rHtt_LTS00343 (advanced sequence CAGAGCTGGTGAACCGTATCC, projected herein as SEQ ID NO: 49; reverse sequence GGCTTAAGCAGGGAGCCAAAA, designed herein as SEQ ID NO: 50; sequence of probe ACTTCATGATGAGCTCGGAGTTCAACX, designed herein as SEQ ID NO:51). The results were calculated as the percentage reduction of huntingtin expression in that of the PBS control and are shown in Table 54. ISIS 388241, ISIS 436684, ISIS 436754, ISIS 437175, ISIS 437507 and ISIS 443139 are each poorly matched by 6 pairs of base or more with the mouse gene sequence (SEQ ID NO: 5) and therefore show no significant inhibition of mouse mRNA levels compared to control. ISIS 419640, ISIS 419641, ISIS 419642, ISIS 436665, ISIS 436668, ISIS 437442, ISIS 444615 and ISIS 444627 each have 1 error with the mouse gene sequence (SEQ ID NO: 5) and show no significant inhibition of levels of rat mRNA compared to control. ISIS 437168 and ISIS 437441 have 2 errors each with the mouse gene sequence (SEQ ID NO: 5) and show no significant inhibition of mouse mRNA levels compared to control. ISIS 436689 and ISIS 444584 have 3 errors each with the mouse gene sequence (SEQ ID NO: 5) and show no significant inhibition of mouse mRNA levels compared to control. Table 54 Percentage reduction in rat huntingtin mRNA levels in rats Example 6: Intracerebroventricular administration of antisense oligonucleotides against huntingtin mRNA - tolerability study in BACHD mice
[000333] Selected compounds were compared to previously engineered compounds ISIS 388241 by ICV administration in BACHD mice.
[000334] Selected compounds plus reference mark 388241 were selected on the basis of systemic and in vitro potency and systemic tolerability as well as CNS potency and tolerability.
[000335] BACHD mice were treated with ISIS oligonucleotides by administering intracerebroventricular (ICV) to a defined mouse brain area in the right lateral ventricle for the purpose of evaluating tolerability of ICV dosage in mice. treatment and surgery
[000336] Groups of five BACHD mice each were administered ISIS 388241, ISIS 437507, ISIS 443139, ISIS 419640, ISIS 419641, ISIS 419642, ISIS 444591, ISIS 436665, ISIS 436671, ISIS 444661, or ISIS 436689 at 150 μg/day released ICV with Alzet 2002 pumps at the rate of 12 μL/day for 2 weeks. A control group of 4 BACHD mice were similarly treated with PBS. Mice were surgically implanted with pumps in the following manner: Mice were individually anesthetized with 3% isoflurane for pump implantation. After two weeks, the mice were again anesthetized and the pump was surgically removed. The animals were then allowed to recover for more than two weeks before being euthanized.
[000337] The mouse body weights were taken weekly during the treatment and recovery periods. After 4 weeks, mice were euthanized using isoflurane and decapitated. The brain was removed for tissue acquisition from the posterior and anterior sections. RNA analysis
Tabela 57 Percentual de redução dos níveis de mRNA de huntingtina de murino em camundongos BACHD por intermédio da administração ICV dos oligonucleotídeos anti-sentido Medição de peso corporal[000338] RNA was extracted from the right hemispheric of the anterior cortex and the posterior cerebellar section of the cannulation site for real-time PCR analysis of huntingtin mRNA levels. Human mutant huntingtin mRNA levels were measured using a series of human RTS2617 primers. Normal mouse huntingtin mRNA levels were measured using a series of murine primer probe RTS2633. Results were calculated as percentage inhibition of human and murine huntingtin mRNA expression compared to control and are presented in Tables 56 and 57 respectively. All antisense oligonucleotides effect significant inhibition of human huntingtin mRNA levels. ISIS 388241, ISIS 437507 and ISIS 443139 are each mismatched by 8 base pairs or more with murine huntingtin mRNA (SEQ ID NO: 3) and therefore show no significant inhibition of murine mRNA levels compared to control . ISIS 444591 has 1 error with murine huntingtin mRNA (SEQ ID NO: 3) and does not show significant inhibition of murine mRNA levels compared to control. ISIS 436689 has 3 errors with murine huntingtin mRNA (SEQ ID NO: 3) and does not show significant inhibition of murine mRNA levels compared to control. Table 56 Percentage reduction in human huntingtin mRNA levels in BACHD mice through ICV administration of antisense oligonucleotides Table 57 Percent reduction in murine huntingtin mRNA levels in BACHD mice by ICV administration of antisense oligonucleotides body weight measurement
[000339] The body weights of mice were measured at baseline and thereafter once a week. The mouse body weights are shown in Table 58 and are expressed as a percentage change in weights taken at baseline. Body weights were considered a measure of the mice's tolerability to ICV administration of the antisense oligonucleotide. 'nd' means there is no data available for that time period. Table 58 Percentage change in body weight of BACHD mice during antisense oligonucleotide treatment mouse survival
[000340] The survival of mice was assessed throughout the entire study period. Table 59 below shows the survival model in the groups of mice treated with ISIS oligonucleotides as well as the control. Table 59 Number of survivals during antisense oligonucleotide treatment Example 7: Inl racerebroventricular administration of antisense oligonucleotides against huntingtin in C57/BL6 mice
[000341] Wild-type C57/BL6 mice were treated with ISIS oligonucleotides by administering intracerebroventricular (ICV) to a defined mouse brain area, in the right lateral ventricle, for the purpose of evaluating the potency of the oligonucleotides against huntingtin from mouse in these mice. treatment and surgery
[000342] Groups of ten C57/BL6 mice each were administered ISIS 408737 (5' TCCTAGTGTTACATTACCGC 3' (SEQ ID NO: 52), 5263 starting site of SEQ ID NO: 3) at 50 µg/day ICV released with Alzet 2002 pumps at the rate of 0.5 µL/day for 7 days or 14 days. A control group of six C57/BL6 mice were similarly treated with PBS. The mice were surgically implanted with the pumps in the following way: Briefly, Alzet osmotic pumps (Model 2002) were assembled according to the manufacturer's instructions. Pumps were filled with a solution containing the antisense oligonucleotide and incubated overnight at 37°C 24 hours before implantation. The animals were anesthetized with 3% isoflurane and placed in a stereotactic frame. After sterilizing the surgical site, a midline incision was made in the head and a subcutaneous pocket was created on the back, where a pre-filled osmotic pump was implanted. A small drill hole was drilled through the head above into the right lateral ventricle. A cannula, connected to the osmotic pump via a plastic catheter, was then placed into the ventricle and glued in place using Loctite adhesive. The incision was closed with sutures. Antisense oligonucleotide or PBS was infused for 7 or 14 days, after which the animals were euthanized according to the human protocol approved by the Institutional Animal Care and Use Committee. Brain and spinal cord tissue were collected and rapidly frozen in liquid nitrogen. Prior to freezing, brain tissue was cut transversely into five sections (S1, S2, S3, S4 and S5) using a mouse brain matrix. Sections 1 to 5 were approximately 2 mm apart from each other with S1 being more rostral and S5 more caudal. RNA and protein analysis
[000343] Total RNA was extracted from mouse brain and spinal cord with RNeasy Protect Mini kit (Qiagen, Mississauga, ON, Canada) for real-time PCR analysis of huntingtin mRNA levels using an RNeasy Mini kit prep (Qiagen). Q-PCR reactions were conducted and analyzed in an ABI Prism 7700 sequence detector (Applied Biosystems). Huntingtin mouse mRNA levels were measured using a murine primer probe ABI # Mm01213820_m1 (Applied Biosystems) and normalized to peptidylpropyl isomerase A mRNA levels. Protein lysates were prepared from mouse brain plates as previously described (Li S.H. and Li X.J., Methods in Molecular Biology (2008), 217:1940-6029). Lysates were run on 3 to 8% tris-acetate gel and transferred using the iBlot dry blotting system (Invitrogen). Blots were probed with anti-beta tubulin (Chemicon) and monoclonal MAB2166 antibody (Millipore) which specifically reacts with murine huntingtin protein. Immunoblots were quantified using Odyssey V3.0 software.
[000344] Results are presented in Table 60 as percent reduction compared to PBS control and demonstrate significant inhibition of huntingtin mRNA and antisense oligonucleotide protein levels on both day 7 and day 14. Table 60 Example 8: Intracerebroventricular administration of antisense oligonucleotides against huntingtin mRNA in cynomolgus monkeys
[000345] Cynomolgus monkeys were treated with ISIS oligonucleotides by administering intracerebroventricular (ICV) to a defined brain area, the lateral ventricles, for the purpose of evaluating oligonucleotide activity in brain tissue against huntingtin mRNA expression. treatment and surgery
[000346] Two groups of 3 cynomolgus monkeys each were administered 0.635 mg/ml (1.5 mg/day) or 1.67 mg/ml (4 mg/day) of ISIS 436689 released ICV with individual ambulatory pumps (Pegasus Vario) at the rate of 0.05 ml/hour for 4 weeks. A control group of 2 cynomolgus monkeys were administered PBS in a similar manner. The groups were administered ISIS 436689 bilaterally. One animal was administered ISIS 436689 at a dosage of 4 mg/day unilaterally to the right ventricle.
[000347] Animals were allowed 10 days to recover from surgery prior to the infusion being performed. During the post-surgery recovery period, animals were maintained on PBS ICV infusion at a flow rate of 0.05 mL/h using an ambulatory infusion pump per ventricle. At the end of the recovery period, each cannula was connected to an individual ambulatory pump (Pegasus Vario) placed inside the primate pouch (Lomir, PJ-02NB). The pumps remain connected until the end of the infusion period. After 4 weeks of administration, the animals were euthanized and the brain, liver and kidneys were collected. htt mRNA RNA analysis
[000348] RNA was extracted from the anterior caudate, posterior caudate, temporal cortex, parietal cortex, hypothalamus, midbrain, hippocampus and spinal cords, as well as the liver and kidney for real-time PCR analysis of mRNA levels of huntingtin. Huntingtin mRNA levels were measured using an RTS2617 human primer probe series and normalized to monkey cyclophilin A levels. Results were calculated as percent inhibition of huntingtin mRNA expression compared to PBS control and are shown in Table 61. ISIS 436689 effected significant inhibition of human huntingtin mRNA levels in the CNS. Table 61 Example 9: Measuring the half-life of ISIS 387898 in the striatum of C57/BL6 mice by single bolus administration
[000349] C57/BL6 mice were administered ISIS 387898 as a simple bolus to the striatum for the purpose of measuring the half-life and duration of action of the antisense oligonucleotide against huntingtin mRNA expression in that tissue. Treatment
[000350] Forty C57/BL6 mice were treated with ISIS 387898 (5' CTCGACTAAAGCAGGATTTC 3' (SEQ ID NO: 53); start position 4042 of SEQ ID NO: 1 and start position 4001 of SEQ ID NO: 3) released as a single 50 μg bolus in a procedure similar to those described in Example 5. Eight C57/BL6 control mice were treated with PBS in a similar procedure. Groups of 4 mice each were euthanized at various time points and striatal tissue extracted in a procedure similar to that described in Example 5. RNA analysis
[000351] RNA was extracted from striatal tissue for real-time PCR analysis of huntingtin mRNA levels. Normal mouse huntingtin mRNA levels were measured using a series of murine primer probe RTS2633. The results are shown in Table 62 and are expressed as percent inhibition compared to the PBS control group on day 7. The inhibitory effect of ISIS 387898 was observed to be prolonged for at least 91 days. Table 62 Effect of ISIS 387898 as single bolus administration on murine huntingtin mRNA expression at various time points in the C57/BL6 striatum Analysis of antisense oligonucleotide concentration in the brain:
[000352] Brain tissues were cut into pieces, weighed, homogenized and extracted using a liquid extraction method - liquid chloroform/phenol. This was followed by solid phase extraction of the supernatant on a phenyl-bonded column before electrokinetic injection of capillary gel electrophoresis. A P/ACE MDQ capillary electrophoresis instrument (Beckman Coulter, Fullerton, CA) was used for the gel-filled capillary electrophoretic analysis. Oligonucleotide peaks were detected by UV absorbance at 260 nm.
Tabela 64 Concentração dependente do tempo de ISIS 387898 no tecido cerebral e seu efeito na expressão htt mRNA como uma porcentagem do controle [000353] The concentration of ISIS 387898 in the brain (μg/g) was plotted against human huntingtin expression as a percentage of the PBS control (Table 63 and Figure 1). The concentration of ISIS 387898 that achieved 50% inhibition of huntingtin mRNA expression (EC50) was calculated. The EC50 was determined to be 0.45 µg/g. The time-dependent concentration of ISIS 387898 in brain tissue and corresponding percentage of huntingtin mRNA expression was also plotted (Table 64 and Figure 2) and the oligonucleotide half-life was calculated as 21 days. Table 63 Table 64 Time-dependent concentration of ISIS 387898 in brain tissue and its effect on htt mRNA expression as a percentage of control Example 10: Measurement of the half-life of ISIS 387898 in the lateral ventricles of BACHD mice by ICV administration
[000354] BACHD mice were administered ISIS 387898 for ICV to the lateral ventricles of the brain for the purpose of measuring the half-life and duration of action of the antisense oligonucleotide against huntingtin mRNA expression in that tissue. Treatment
[000355] Twenty-eight BACHD mice were treated with ISIS 387898 delivered by ICV administration at 75 μg/day for 2 weeks in a procedure similar to that described in Example 9. Twenty-eight BACHD control mice were treated with PBS in a similar procedure that described in Example 9. Groups of 4 mice each from both the treatment and control groups were euthanized at the time points biweekly and anterior cortical tissue extracted in a procedure similar to that described in Example 9. RNA analysis
Tabela 66 Efeito de ISIS 387898 administrado ICV na expressão de mRNA de huntingtina de murino em vários pontos de tempo [000356] RNA was extracted from the right hemisphere, both anterior and posterior to the cannulation site for real-time PCR analysis of huntingtin mRNA levels. Human mutant huntingtin mRNA levels were measured using a series of human RTS2617 primers. Normal mouse huntingtin mRNA levels were measured using a series of murine primer probe RTS2633. Human mutant huntingtin mRNA expression levels are shown in Table 65 and are expressed as percent inhibition compared to the mean of that measured in the PBS control groups. Normal murine huntingtin mRNA expression levels are shown in Table 66 and are expressed as percent inhibition compared to the mean of that measured in the PBS control groups. The inhibitory effect of ISIS 387898 was observed to be prolonged for 91 days. Table 65 Table 66 Effect of ISIS 387898 administered ICV on murine huntingtin mRNA expression at various time points Analysis of antisense oligonucleotide concentration in the brain:
Tabela 68 Concentração dependente do tempo de ISIS 387898 no tecido cerebral e seu efeito na expressão htt mRNA como uma porcentagem do controle [000357] Brain tissue was processed in a procedure similar to that described in Example 9. The concentration of ISIS 387898 in the forebrain cortex (μg/g) was plotted against inhibition of human huntingtin as a percentage of the PBS control (Table 67 and figure 3) and the EC50 was calculated to be 26.4 μg/g. The time-dependent concentration of ISIS 387898 in brain tissue was also plotted (Table 68 and Figure 4) and the oligonucleotide half-life was calculated as 21 days. Table 67 Table 68 Time-dependent concentration of ISIS 387898 in brain tissue and its effect on htt mRNA expression as a percentage of control Example 11: Measurement of the half-life of ISIS 388241 and ISIS 443139 in the lateral ventricles of BACHD mice by ICV administration
[000358] BACHD mice were administered ISIS 388241 or ISIS 443139 by ICV to the lateral ventricles of the brain for the purpose of measuring the half-life and duration of action of the antisense oligonucleotide against huntingtin mRNA expression in that tissue. Treatment
[000359] Twenty BACHD mice were treated with ISIS 38241 released by ICV administration at 50 μg/day for 2 weeks in a procedure similar to that described in Example 9. Twenty BACHD mice were treated with ISIS 443139 released by ICV administration at 50 μg/day for 2 weeks in a procedure similar to that described in Example 9. Twenty BACHD control mice were treated with PBS in a procedure similar to that described in Example 9. Groups of 4 mice each from both the treatment and control groups were subjected euthanized at the time points biweekly and tissue extracted in a procedure similar to that described in Example 9. RNA analysis
[000360] RNA was extracted from the right hemisphere, both anterior and posterior to the cannulation site for real-time PCR analysis of huntingtin mRNA levels. Human mutant huntingtin mRNA levels were measured using a series of human RTS2617 primers. The results are shown in Table 69 and are expressed as percent inhibition compared to the mean of that measured in the PBS control groups. The inhibitory effects of both ISIS 388241 and ISIS 443139 have been observed to be prolonged for at least 16 weeks.
[000361] Both ISIS 388241 and its mixed main-chain equivalent, ISIS 443139, have more than 3 errors with murine huntingtin mRNA (SEQ ID NO: 5) and therefore show no significant inhibition of compared murine mRNA levels to control. Table 69 Effect of ISIS 388241 and ISIS 443139 administered ICV on human huntingtin mRNA expression at various time points Analysis of antisense oligonucleotide concentration in the brain:
Tabela 71 Concentração de ISIS 443139 no tecido cerebral e seu efeito na expressão htt mRNA como uma porcentagem do controle [000362] Brain tissue was processed in a procedure similar to that described in Example 9. The time-dependent concentration of ISIS 388241 in posterior brain tissue was plotted (Table 70 and Figure 5) and the oligonucleotide half-life was calculated as 20 days . The time-dependent concentration of ISIS 443139 in posterior brain tissue was plotted (Table 71 and Figure 6) and the oligonucleotide half-life was calculated as 20 days. Table 70 Concentration of ISIS 384241 in brain tissue and its effect on htt mRNA expression as a percentage of control Table 71 Concentration of ISIS 443139 in brain tissue and its effect on htt mRNA expression as a percentage of control Example 12: Effect of antisense inhibition of mutant human huntingtin on the motor performance of BACHD mice
[000363] BACHD mice were treated with ISIS oligonucleotides through intracerebroventricular (ICV) administration for the purpose of evaluating the effect of oligonucleotides against huntingtin mRNA expression on their motor performance by means of the rotarod assay. Treatment
[000364] The acceleration of the rotarod test was carried out on the Ugo Basile rotarod. Animals were placed on the rotarod at a speed of 2 RPM, the rotarod accelerated to 40 RPM in 5 minutes. The duration until the drop is recorded. The duration until the fall is defined by the animal that falls from the rotarod, or that stops walking, leaning on the rotarod and rotating it. 6-month-old BACHD mice and their non-transgenic litters were trained to run on the rotarod for one week before treatment. This consists of three consecutive trials of 5 minutes each, with a period of 20 minutes remaining between trials. A group of 15 BACHD mice were then treated with ISIS 388241 at 50 μg/day ICV released with Alzet 2002 pumps at the rate of 12 μL/day for 2 weeks. The mice were surgically implanted with the pumps in a similar procedure as that described in Example 6. A control group of 14 BACHD mice were treated with PBS in a similar manner. A control group of 9 non-transgenic litters were treated with PBS in a similar way. Rotarod performance test
[000365] At the end of the treatment period, the pumps were removed and two weeks later, the first post-treatment rotarod was conducted. Rotarod behavior was analyzed monthly until the mice were 11 months old. Each month, animals were placed on the rotarod for three trials performed on the day for 2 days. The results are shown in Fig. 7 as well as in Table 72 expressed as a duration to drop in seconds. Baseline values at 6 months of age were taken prior to treatment and the time points given are the age of the mice in which the trial was conducted. The data indicate that treatment of BACHD mice with ISIS 388241 increases the duration to fall compared to that seen in untreated BACHD mice. Table 72 Effect of antisense inhibition of mutant huntingtin mRNA on a duration to fall (sec) Example 13: Effect of antisense inhibition of mutant human huntingtin and wild-type murine huntingtin mRNA on motor performance of BACHD mice
[000366] BACHD mice were treated with ISIS oligonucleotides through intracerebroventricular (ICV) administration for the purpose of evaluating the effect of oligonucleotides against huntingtin mRNA expression on their motor performance by means of the rotarod assay. Treatment
[000367] The acceleration of the rotarod test was carried out on the Ugo Basile rotarod. Animals were placed on the rotarod at a speed of 2 RPM, the rotarod accelerated to 40 RPM in 5 minutes. The duration until the fall was recorded. The duration until the fall is defined by the animal that falls from the rotarod or that stops running in the rotarod, that leans in the rotarod and rotates it. Two-month-old BACHD mice and their non-transgenic litters were trained to run on the rotarod for one week before treatment. This consists of three consecutive trials of 5 minutes each, with a 20-minute rest period between trials. Groups of 17-21 BACHD mice each were then treated with ISIS 388241 at 50 μg/day, ISIS 408737 at 75 μg/day, or ISIS 387898 at 75 μg/day, released with ICV 2002 Alzet pumps at a rate of 0. 5 μL/hour for 2 weeks. The mice were surgically implanted with the pumps in a procedure similar to those described in Example 6. A control group of 20 BACHD mice were treated with PBS in a similar manner. Control mice from non-transgenic groups were also similarly treated with ISIS or PBS oligonucleotides in a similar manner. Rotarod performance test
[000368] At the end of the treatment period, the pumps were removed and two weeks later, the first post-treatment rotarod was conducted. Rotarod behavior was analyzed monthly until the mice were 10 months old. Each month, animals were placed on the rotarod for 3-5 trials performed on the day for 3 consecutive days. The results are shown in Table 73 expressed as a duration to drop in seconds. Baseline values at 2 months of age were taken prior to treatment and the time points given are the age of the mice in which the trial was conducted. ISIS 387898 (designed in the table as mouse-human ASO) is cross-reactive for both mouse and human huntingtin mRNA and the refore should inhibit both mutant human huntingtin mRNA and wild-type murine huntingtin mRNA in mice. ISIS 388241 (designed in the table as human ASO) specifically targets human huntingtin mRNA and is poorly matched by 8 base pairs with murine huntingtin mRNA. Therefore, ISIS 388241 will specifically inhibit only mutant human huntingtin mRNA and not wild-type murine huntingtin mRNA in mice. ISIS 408737 (designed in the table as mouse ASO) specifically targets murine huntingtin mRNA and is mismatched by 7 base pairs with human huntingtin mRNA. Therefore, ISIS 408737 will specifically inhibit only wild-type murine huntingtin mRNA and not mutant human huntingtin mRNA in mice. 'Tg' indicates BACHD mice and 'non-Tg' indicates non-transgenic control mice.
[000369] The results of the study indicate that inhibition of mutant human huntingtin mRNA by ISIS 388241 (human Tg-ASO) significantly improves the performance of mice in the rotarod assay compared to control (Tg-PBS). The results also indicate that treatment of mice with ISIS 387898 (Tg-human-mouse ASO), which targets both huntingtin mRNA in mutant and wild-type mice, does not cause any harmful effects on the mice's motor performance and, in fact, also significantly improved rotarod performance compared to control (Tg-PBS). Mice treated with ISIS 408737 (Tg-mouse ASO) do not show improved rotarod performance compared to PBS control, as expected, as the oligonucleotide does not target the mutant huntingtin mRNA. Non-transgenic controls were used as positive controls in this assay. Table 73 Effect of huntingtin mRNA antisense inhibition on duration to fall (sec) Example 14: Effect of huntingtin mRNA antisense inhibition on the brain mass of R6/2 mice
[000370] R6/2 mice were treated with ISIS oligonucleotides through intracerebroventricular (ICV) administration for the purpose of evaluating the effect of oligonucleotides against huntingtin mRNA expression on brain weight and volume. Treatment
[000371] R6/2 mice were housed in groups of up to 5 per cage (mixed genotypes, single sex). All mice were housed in shoebox cages with sterile wooden forage coating the development of that and were moved as often as necessary to provide the animals with dry forage. This basic environment has been enriched with the addition of fun tunnels, patchwork nest and plastic bones for all mice; that is, an environmentally enriched cage containing a mouse tunnel, (amber, certified, clear, BioServ product # K3323), a Petite Green Gumabone (BioServ product # K3214), and a nest (Hockley et al., Ann Neurol. 2002, 51: 235-242). Food and water were available ad libitum to the mice in their home cages.
[000372] A group of ten six-month-old R6/2 mice were administered 50 μg/day of ISIS 388817 released ICV with Alzet 1004 pumps at the rate of 0.12 μl/hour for 4 weeks. A group of two non-transgenic litters were administered 50 µg/day of released ISIS 388817 in a similar manner. A control group of five R6/2 mice were administered 50 μg/day of ISIS 141923 released in a similar manner. A control group of nine R6/2 mice were administered PBS released in a similar manner. A group of eight non-transgenic litters were administered PBS released in a similar manner. A group of four untreated eight-week-old pre-symptomatic R6/2 were also included in the study. brain weight measurement
Exemplo 15: Efeito da inibição anti-sentido de mRNA de huntingtina no desempenho de ansiedade de camundongos YAC128[000373] The animals were anesthetized with isoflurane and then subjected to transcardial perfusion with ice-cold Sorenson phosphate buffer (SPB) and fixed with 4% paraformaldehyde in SPB. Brains were removed and prepared with coronal sections immediately rostral to the forebrain (removing the olfactory bulbs) and immediately caudal to the cerebellum (removing the spinal cord). The remaining brain was weighed in mg. The results are shown in Fig. 8 and Table 74 and demonstrate the increase in brain weight in R6/2 mice treated with ISIS 388817 compared to control PBS. Table 74 Effect of mutant huntingtin mRNA antisense inhibition on brain weight (mg ) Example 15: Effect of huntingtin mRNA antisense inhibition on anxiety performance of YAC128 mice
[000374] YAC128 mice were treated with ISIS oligonucleotides via intracerebroventricular (ICV) administration for the purpose of evaluating the effect of oligonucleotides against huntingtin mRNA expression on anxiety in these mice as measured by their performance in the opening and high field more labyrinth tests. Treatment
[000375] A group of seven five-month-old YAC128 mice were administered 50 μg/day of ISIS 388241 released ICV with Alzet 1004 pumps at the rate of 0.5 μl/hour for 14 days. A control group of four YAC128 mice were similarly treated with PBS. A control group of eight non-transgenic FVB/NJ litters were included in the study and did not receive any treatment. The mice were surgically implanted with the pumps in the following way: Briefly, Alzet osmotic pumps (Model 2002) were assembled according to the manufacturer's instructions. Pumps were filled with a solution containing the antisense oligonucleotide and incubated overnight at 37°C 24 hours before implantation. The animals were anesthetized with 3% isoflurane and placed in a stereotactic frame. After sterilizing the surgical site, a midline incision was made in the head and a subcutaneous pocket was created on the back, where a pre-filled osmotic pump was implanted. A small drill hole was drilled through the head above into the right lateral ventricle. A cannula, connected to the osmotic pump via a plastic catheter, was then placed into the ventricle and glued in place using Loctite adhesive. The incision was closed with sutures. Antisense oligonucleotide or PBS was infused for 14 days, after which the pumps were removed. Animals were allowed to recover for 2 weeks after behavioral analysis was done and mice were finally euthanized according to the human protocol approved by the Institutional Animal Care and Use Committee. Brain and spinal cord tissue were collected and rapidly frozen in liquid nitrogen. Prior to freezing, brain tissue was cut transversely into five sections (S1, S2, S3, S4 and S5) using a mouse brain matrix. Sections 1 to 5 were approximately 2 mm apart from each other with S1 being more rostral and S5 more caudal. opening field test
[000376] Mice were placed in an opening field arena (Med Associates) using photobeam breaks to measure horizontal and vertical movement in a 30-minute test session. Data was analyzed using Activity Monitor software to examine total ambulatory movement within the arena and movement within the center of the arena as a measure of anxiety. The YAC128 control mice were expected to spend less time in the center of the arena compared to their less anxiety-prone, non-transgenic FVB/NJ litter. The results are shown in Fig. 9 and Table 75 and indicate that treatment of YAC128 mice with decreased antisense oligonucleotide anxiety in these mice, according to the parameters of the opening field assay. Table 75 Effect of antisense inhibition of mutant htt mRNA on opening field performance of YAC128 mice
[000377] The mechanism consisting of two open arms and two closed arms each measuring 65 x 6.25 cm and 50 cm high above the ground. The mice were placed in the center of the mechanism and their location was recorded in a 5-minute test session. The YAC128 control mice were expected to spend less time in the mechanism's open arms compared to their less anxiety-prone, non-transgenic FVB/NJ litters. The results are shown in Fig. 10 and Table 76 and indicate that the treatment of YAC128 mice with anxiety decreased by the antisense oligonucleotide in these mice, according to the parameters of the elevated plus maze assay. Table 76
[000378] Total RNA was extracted from mouse brain and spinal cord with RNeasy Protect Mini kit (Qiagen, Mississauga, ON, Canada) for real-time PCR analysis of huntingtin mRNA levels using an RNeasy Mini kit prep (Qiagen). Q-PCR reactions were conducted and analyzed in an ABI Prism 7700 sequence detector (Applied Biosystems). Human huntingtin mRNA levels were measured using an RTS2686 human primer probe series and normalized to peptidylpropyl isomerase A mRNA levels.
[000379] Protein lysates were prepared from mouse brain plates as previously described (Li S.H. and Li X.J., Methods in Molecular Biology (2008), 217:1940-6029). Lysates were run on 3 to 8% tris-acetate gel and transferred using the iBlot dry blotting system (Invitrogen). Blots were probed with anti-beta tubulin (Chemicon) and mouse monoclonal EM48 antibody that specifically reacts with human huntingtin protein (Millipore). Immunoblots were quantified using Odyssey Software V3.0.
[000380] Results are shown in Table 77 as percent reduction compared to PBS control and demonstrate significant inhibition of huntingtin mRNA and antisense oligonucleotide protein levels. Table 77 Example 16: Intracerebroventricular administration of antisense oligonucleotides against huntingtin in C57/BL6 mice
[000381] C57/BL6 mice were treated with ISIS oligonucleotides through the administration of intracerebroventricular (ICV) in the right lateral ventricle, for the purpose of evaluating the tolerability of oligonucleotides in these mice. treatment and surgery
[000382] Groups of five C57/BL6 mice each were administered ISIS 387916, ISIS 437527, ISIS 444578, ISIS 444584, ISIS 444607, ISIS 444608, ISIS 444627, ISIS 444652, ISIS 444659, ISIS 444660, or ISIS 444661 at 150 μg /day ICV released with Alzet 2002 pumps at the rate of 0.5 μL/day for 2 weeks. A control group of six C57/BL6 mice were similarly treated with PBS. The procedure for implanting the pumps and administering the oligonucleotide is described in Example 6.
[000383] Animals were allowed to recover for two weeks before being euthanized using isoflurane. Brain and spinal cord tissue were collected and rapidly frozen in liquid nitrogen. Prior to freezing, brain tissue was cut transversely into five sections (S1, S2, S3, S4 and S5) using a mouse brain matrix. Sections 1 to 5 were approximately 2 mm apart from each other with S1 being more rostral and S5 more caudal. RNA analysis
[000384] Total RNA was extracted from the anterior and posterior cortices of the brain for real-time PCR analysis of huntingtin mRNA levels using an RNeasy Mini prep kit (Qiagen). RT-PCR reactions were conducted on an ABI Prism 7700 sequence detector (Applied Biosystems). Huntingtin mouse mRNA levels were measured using a series of murine primer probe RTS2633 and normalized to cyclophilin mRNA levels. Results are shown in Table 78 as percent reduction compared to PBS control. ISIS 387916, ISIS 437527, ISIS 444627 and ISIS 444652 all have an error with the murine huntingtin mRNA (SEQ ID NO: 3) and therefore do not show significant inhibition of murine mRNA levels compared to the control.
Tabela 79 Aumento do percentual na expressão AIF1 mRNA comparado ao controle em camundongos C57/BL6 Medição de pesos corporais[000385] The microglial marker, AIF1 was also measured by RT-PCR analysis using a series of murine primer probe mAIF1_LTS00328 (advanced sequence TGGTCCCCCAGCCAAGA, projected in this as SEQ ID NO: 54; reverse sequence CCCACCGTGTGACATCCA, designed in this as SEQ ID NO. °: 55; probe sequence AGCTATCTCCGAGCTGCCCTGATTGG, projected herein as SEQ ID NO: 56). The results are shown in Table 79 and indicate that the tested ISIS oligonucleotides do not induce an inflammatory response. Table 78 Percentage inhibition of murine huntingtin mRNA compared to control in C57/BL6 mice Table 79 Percentage increase in AIF1 mRNA expression compared to control in C57/BL6 mice body weight measurement
[000386] Body weights were measured at regulatory intervals throughout the study period and are shown in Table 80. These weights were used as an indicator of tolerability. The mice treated with ISIS 437527, ISIS 444584, and ISIS 444652 have consistent body weight throughout the study period and were considered the most tolerable of all the ISIS oligonucleotides included in the study. 'n/a' does not indicate the data by which the group of mice. Table 80 Body weights of C57/BL6 mice after antisense oligonucleotide treatment Example 17: Assay for neurotoxic effects of bolus administration of antisense oligonucleotides in rat striatal tissue.
[000387] Sprague-Dawley rats were treated with ISIS oligonucleotides via bolus administration to the striatum for the purpose of evaluation for the introduction of the microglial marker AIF1 as a measure of CNS toxicity. treatment and surgery
[000388] Groups of four Sprague-Dawley Rats were administered ISIS 388241, ISIS 443139, ISIS 436671, ISIS 437527, ISIS 444584, ISIS 444591, or ISIS 444591, or ISIS 444652 released as a single cake at a concentration of 25 μg, 50 μg, 75 µg, or 100 µg.
[000389] A group of 4 rats were similarly treated with ISIS 387916, delivered as a simple bolus at 10 μg, 25 μg, 50 μg, or 75 μg concentrations. A control group of 4 mice were similarly treated with PBS. Seven days after bolus administration, rats were euthanized using isoflurane and organs were removed. The animals were decapitated and the brain was removed for striatal tissue dissection. A pair of thin curved forceps were placed directly into the brain anterior to the hippocampus to make a transverse incision in the cortex and underlying tissues by direct dissection. The ends of another pair of thin curved forceps were placed directly along the mid-sagittal sinus midway between the hippocampus and the olfactory bulb to make a longitudinal incision, cutting the corpus callus by direct dissection. The first pair of forceps was then used to re-reflect the resulting corner of the cortex that exposes the striatum and the inner capsule and then dissect the inner capsule again and again from the striatum. The second series of forceps was placed such that the curved ends were in place of the striatum and compressed to isolate tissue. The first series of forceps was used to arrest the posterior end of the striatum and to remove the striatum from the brain. RNA analysis of AIF1 expression levels
[000390] RNA was extracted from striatal tissue for real-time PCR analysis of AIF1 mRNA levels. Rat AIF1 levels were measured using the rAif1_LTS00219 rat primer probe series. The results were calculated as the percentage of AIF1 expression in that of the PBS control and are shown in Table 81. The results indicate that ISIS 388241, ISIS 443139, ISIS 436671, ISIS 444591, ISIS 437527, ISIS 444584 and ISIS 444652 were well tolerated in the brain of the mouse. Table 81 Percent expression of AIF1 mRNA levels in vivo as a measure of neurotoxicity RNA analysis of huntingtin expression levels
[000391] RNA was extracted from striatal tissue for real-time PCR analysis of huntingtin mRNA levels. Rat huntingtin mRNA levels were measured using the rHtt_LTS00343 rat primer probe series. The results were calculated as the percent reduction in huntingtin expression in that of the PBS control and are shown in Table 82. ISIS 388241 and ISIS 443139 are each mismatched by 6 base pairs or more with the mouse gene sequence (SEQ). ID No: 5) and therefore do not show significant inhibition of rat mRNA levels compared to control. ISIS 444584 has 3 errors with the mouse gene sequence (SEQ ID NO: 5) and therefore does not show significant inhibition of mouse mRNA levels compared to control. Table 82 Percent reduction in rat huntingtin mRNA levels in rats Example 18: Dosage-dependent antisense inhibition of huntingtin mRNA in primary cynomolgus hepatocytes
[000392] ISIS 437527, ISIS 444584 and ISIS 444652 were tested on cynomolgus primary hepatocytes at various dosages. The reference tag oligonucleotides, ISIS 387916 and ISIS 388241 were also included for comparison. Cells were placed at a density of 35,000 cells per well and transfected using electroporation at 39,0625 nM, 78,125 nM, 156.25 nM, 312.5 nM, 625 nM, 1,250 nM, 2,500 nM, 5,000 nM, 10,000 nM and 20,000 nM concentrations of each antisense oligonucleotide. After approximately 16 hours, RNA was isolated from the cells and huntingtin mRNA transcript levels were measured by quantitative real-time PCR using the RTS2686 primer series. Huntingtin mRNA transcript levels were normalized to a total RNA content as measured by RIBOGREEN®. The results are shown in Table 83 as percentage inhibition of huntingtin relative to untreated control cells. The control oligonucleotide, ISIS 141923 was included in this assay and does not demonstrate huntingtin mRNA inhibition as expected.
[000393] ISIS 437527, ISIS 444584, and ISIS 444652 have lower IC50 values than the reference tag oligonucleotide, ISIS 388241. ISIS 437527 and ISIS 444652 had lower IC50 values than the benchmark oligonucleotide, ISIS 387916 Table 83 Dose-dependent antisense inhibition of huntingtin mRNA in primary cynomolgus hepatocytes Example 19: Measurement of the half-life of ISIS oligonucleotides in BACHD mice by single intrastriatal bolus administration
[000394] BACHD mice were administered ISIS oligonucleotides as a simple bolus to the striatum for the purpose of measuring the duration of action of antisense oligonucleotides against huntingtin mRNA expression, or its half-life, in that tissue. treatment and surgery
[000395] Groups of 25 BACD mice each were treated with ISIS 388241, ISIS 436689, ISIS 436671, or ISIS 444591, delivered as a single 40 μg bolus in a procedure similar to that described in Example 4. A control group of 25 BACHD mice were treated with PBS in a similar procedure. At various time points, 5 mice from each group were euthanized and striatal tissue was extracted. A pair of thin curved forceps was placed in the brain just before the hippocampus to make a transverse incision in the cortex and underlying tissues by direct dissection. The ends of another pair of thin curved forceps were placed along the mid-sagittal sinus midway between the hippocampus and the olfactory bulb to make a longitudinal incision, cutting through the body callus by direct dissection. The first pair of forceps was then used to re-reflect the corner of the cortex that exposes the striatum and the inner capsule and then to dissect the inner capsule again from the striatum. The second series of forceps was placed such that the ends curved into the side of the striatum and compressed to isolate the tissue. The first series of forceps was used to arrest the posterior end of the striatum and to remove the striatum from the brain. RNA analysis
Tabela 85 Tabela 86 Vida média de oligonucleotídeos ISIS na seção anterior do cérebro em camundongos BACHD após injeção de bolo intraestriatal Medição de pesos corporais[000396] RNA was extracted from the anterior and posterior sections of striatal tissue for real-time PCR analysis of huntingtin mRNA levels. Human mutant huntingtin mRNA levels were measured using RTS2617. Normal mouse huntingtin mRNA levels were measured using a series of murine primer probe RTS2633. The results are shown in Tables 84 and 85 and are expressed as percent inhibition compared to the mean of the PBS control group at week 1, week 10 and week 20. The half-life of ISIS oligonucleotides in the anterior brain section was calculated from the inhibition data and is shown in Table 86. Table 84 Table 85 Table 86 Half-life of ISIS oligonucleotides in the anterior section of the brain in BACHD mice after intrastriatal bolus injection body weight measurement
Exemplo 20: Efeito de administração intratecal de ISIS 437527 em ratos Sprague Dawley[000397] Body weights were measured at regular intervals and are presented in Table 87 as a percentage of the mice's weight at baseline. These weights were used as an indicator of tolerability. There is no adverse change in body weight in any of the mice treated with ISIS oligonucleotides. Table 87 Percentage change in body weight of BACHD mice after antisense oligonucleotide treatment Example 20: Effect of intrathecal administration of ISIS 437527 in Sprague Dawley rats
[000398] Sprague Dawley rats were dosed with ISIS 437527 by intrathecal (IT) administration as a single dose, repeated dose or continuous infusion. treatment and surgery
[000399] Rats were anesthetized with isoflurane and a 28-gauge polyurethane catheter was placed in the IT lumbar space of each rat. The proximal end of the catheter was connected to a dosage pedestal that was extended through the skin by animals in the groups receiving the bolus injections. The catheter for animals in the group receiving continuous infusion was connected to an ALZET pump (Model 2ML1) that was placed in a subcutaneous pocket on the dorsal aspect of each animal. Post-surgically, the animals receive a simple intramuscular dose of ceftiofur sodium (5 mg/kg) and butorphanol tartrate (0.05 mg/kg). Rats receiving continuous infusion begin to receive oligonucleotide dosing immediately. Animals that received the bolus injections were allowed a surgical recovery period of at least five days after which catheter potency was assessed.
[000400] A group of 5 Sprague Dawley rats were given a 350 µg single bolus injection of intrathecally released ISIS 437527. Another group of 5 Sprague Dawley rats were administered by bolus injections of 120 µg of intrathecally released ISIS 437527 three times during the 1 week course. Another group of 5 Sprague Dawley rats were administered by bolus injections of 350 µg of intrathecally released ISIS 437527 three times over the course of 1 week. Another group of 5 Sprague Dawley rats were administered 50 μg/day of ISIS 437527 delivered by continuous infusion at a rate of 0.01 mL/hour for 7 days. A control group of 5 Sprague Dawley rats was administered by injections of intrathecally released PBS bolus three times during the 1 week course. Each group was given a 7-day recovery period, after which the rats were euthanized. The brain and spinal cord from all groups and were collected and analyzed. RNA analysis of huntingtin expression levels
Análise de RNA dos níveis de expressão AIF1[000401] RNA was extracted from frontal cortex, temporal cortex and cervical medulla for real-time PCR analysis of huntingtin mRNA levels. Rat huntingtin mRNA levels were measured using the rHtt_LTS00343 primer probe series normalized to Cyclophilin levels. Results are shown in Table 88 and are expressed as percent inhibition compared to the mean of the PBS control groups. Table 88 Percent inhibition of huntingtin mRNA expression in Sprague Dawley rats RNA analysis of AIF1 expression levels
Exemplo 21: Medição da vida média de ISIS 436689 nos tecidos SNC dos macacos cinomólgos por intermédio da administração intratecal[000402] RNA was extracted from the frontal cortex, temporal cortex and cervical medulla for real-time PCR analysis of AIF1 mRNA levels. Rat AIF1 levels were measured using the rAif1_LTS00219 rat primer probe series. The results were calculated as the percentage of AIF1 expression in that of the PBS control and are shown in Table 89. The results indicate that repeated IT bolus administration leads to inflammation of the cervical cord tissues. Continuous IT administration and single IT bolus administration were well tolerated in rats. Table 89 Percent Expression of AIF1 mRNA Levels in Sprague Dawley Rats as a Measure of Neurotoxicity Example 21: Measurement of the half-life of ISIS 436689 in CNS tissues of cynomolgus monkeys via intrathecal administration
[000403] Cynomolgus monkeys were administered ISIS 436689 intrathecally (IT) for the purpose of measuring the half-life and duration of action of the antisense oligonucleotide against huntingtin mRNA expression in various CNS tissues. Treatment
[000404] The study was conducted at Northern Biomedical Research, MI. Before starting treatment, the monkeys were kept in quarantine for a period of time of 4 weeks, during which standard serum chemistry and hematology panels, examination of faecal samples for egg and parasites, and a tuberculosis test were conducted. to evaluate abnormal or diseased monkeys. The monkeys were implanted with intrathecal lumbar catheters using polyurethane catheters connected to a subcutaneous titanium port (titanium/plastic P.A.S. PORT® Elite port with Ultra lock connector). For continuous infusion using an external pump, animals were anesthetized to connect the dosing mechanism to the port. The animals were pretreated with atropine sulfate by subcutaneous injection at a dosage of 0.04 mg/kg. Approximately 15 minutes later, an intramuscular dosage of 8 mg/kg of ketamine HCl was administered to induce sedation. Animals were masked in a surgical anesthesia plan, intubated and maintained on approximately 1 L/min oxygen and 2% halothane or isoflurane. Animals received a single intramuscular dose of 5 mg/kg of ceftiofur sodium antibiotic. An incision was made near the modified needle holder placement port. The modified needle was placed in the port and secured with sutures. On recovery from surgery, a bag was placed in the animal.
[000405] Fifteen male cynomolgus monkeys were administered 4 mg/day of ISIS 436689 at a concentration of 1.67 mg/ml and at a flow rate of 2.4 ml/day for 21 days. A control group of 3 cynomolgus monkeys was administered PBS in a similar manner for the same length of time. Groups of 3 monkeys each were left in the recovery period of 1 day, 2 weeks, 4 weeks, or 8 weeks, after which they were euthanized. During the study period, the monkeys were observed daily for signs of illness or distress.
[000406] All animals were sedated with an intramuscular injection of 8.0 mg/kg of ketamine HCl, maintained in a halothane or isoflurane/oxygen mixture, and provided with an intravenous bolus of Na heparin at 200 IU/kg. The animals were perfused through the left cardiac ventricle with 0.001 % sodium nitrite in saline solution.
[000407] At the time of sacrifice, the brain was cut into a 3 mm thick coronal slice brain matrix. Various structures of the brain were sampled using a 4 mm biopsy. A 4 mm diameter sample from each structure was placed in 2 mL screw cap tubes containing 1.0 mL RNAlater RNA Stabilization Solution (Qiagen, CA), incubated for 1 hour at room temperature and then frozen. Adjacent 6 mm diameter samples were placed in 2 mL screw cap tubes and frozen for pharmacokinetic analysis.
[000408] The spinal cord was dissected into the cervical, thoracic and lumbar sections and approximately 3mm thick sections of each area of the spinal cord were taken by RNA and pharmacokinetic analysis. These samples were processed in a manner similar to that of brain samples.
[000409] Liver samples were collected by pharmacokinetic and RNA analysis. Samples were processed in a manner similar to those from the brain and spinal cord as described above. RNA analysis
Tabela 91 Efeito de ISIS 436689 administrado IT na expressão de mRNA de huntingtina em vários tecidos cerebrais em vários pontos de tempo Medição da concentração de oligonucleotídeo por ELISA[000410] RNA was extracted from lumbar spinal cord, thoracic spinal cord, cervical spinal cord, frontal cortex, occipital cortex, cerebellar cortex, caudate tissue, hippocampus, midbrain and pons for real-time PCR analysis of levels of huntingtin mRNA with RTS2617 Primer Probe Set. Results measured in the various sections of the spinal cord are shown in Table 90 and are expressed as percent inhibition compared to that measured in the PBS control group at 8 weeks. Results measured in various brain sections are shown in Table 91 and are expressed as percent inhibition compared to that measured in the PBS control group at 8 weeks. Table 90 Effect of ISIS 436689 administered IT on huntingtin mRNA expression in spinal cord at various time points Table 91 Effect of ISIS 436689 administered IT on huntingtin mRNA expression in various brain tissues at various time points Measurement of oligonucleotide concentration by ELISA
[000411] Tissues (20 mg) were cut into pieces, weighed and homogenized before liquid/liquid extraction using phenol/chloroform. The supernatant was removed, lyophilized and reconstituted in human EDTA plasma (1 ml) before being analyzed using an ELISA hybridization procedure.
Tabela 93 Vida média de ISIS 436689 administrado IT na expressão de mRNA de huntingtina em vários tecidos [000412] ISIS 436689 was detected in tissues by hybridization to a labeled complementary cut probe (digoxigenin at the 5' end and a C18 spacer and BioTEG at the 3' end). The complex was then captured on a neutrovidin coated plate and S1 nuclease was added to digest the unhybridized cut-off probes. Since ISIS 436689 protected the cutting probe from digestion, the undigested cutting probe was used as a measure of oligonucleotide concentration. The undigested cut probe was digested using an alkaline phosphatase-conjugated anti-digoxigenin antibody by reading fluorogenic substrate. Oligonucleotide concentrations were measured in the cervical, thoracic and lumbar sections of the spinal cord and in the liver on days 7, 20, 34 and 62 of the recovery period and are shown in Table 92. The half-life of ISIS 436689 in these tissues was calculated at from these data and is presented in Table 93. The data indicate that the oligonucleotide was mainly concentrated in the CNS with negligible concentrations in the systemic tissues. Table 92 Table 93 Half-life of ISIS 436689 administered IT on huntingtin mRNA expression in various tissues

权利要求:
Claims (10)
[0001]
1. Modified single-stranded oligonucleotide targeting a huntingtin-encoding nucleic acid capable of inhibiting huntingtin expression, characterized by the fact that it comprises: a slack segment consisting of linked deoxynucleotides; a 5' wing segment consisting of linked nucleosides; and a 3'-wing segment consisting of linked nucleosides; wherein the slack segment is positioned between the 5'-wing segment and the 3'-wing segment, wherein each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar; wherein the nucleoside bonds within the slack segment, the bonds connecting the slack segment to the 5' or 3' wing segment, and the bonds to the 5' and 3' majority nucleosides of each wing segment are all phosphorothioate linkages, the nucleoside linkages connecting the rest of the nucleosides from both the 5' and 3' wing segments and phosphodiester linkages; and wherein all cytosines are 5-methylcytosines; and wherein the nucleobase sequence of the oligonucleotide consists of a sequence according to SEQ ID NO: 22 or 32.
[0002]
2. Oligonucleotide according to claim 1, characterized in that the nucleobase sequence of the oligonucleotide consists of the sequence according to SEQ ID NO: 22 (ISIS 443139).
[0003]
3. Oligonucleotide according to claim 1, characterized in that the oligonucleotide nucleobase sequence consists of the sequence according to SEQ ID NO: 32 (ISIS 444652).
[0004]
4. Oligonucleotide, according to any one of claims 1 to 3, characterized in that the oligonucleotide is conjugated.
[0005]
5. Composition, characterized in that it comprises the oligonucleotide, as defined in any one of claims 1 to 4, or a salt thereof and at least one of a pharmaceutically acceptable carrier or diluent.
[0006]
6. Composition according to claim 5, characterized in that it is for use in therapy.
[0007]
7. Use of an oligonucleotide as defined in any one of claims 1 to 4, or a salt thereof, characterized in that it is in the preparation of a composition to prevent, treat, ameliorate, and reduce the rate of progression of the disease of Huntington in an animal.
[0008]
8. Use according to claim 7, characterized in that the animal is a human.
[0009]
9. Use according to claim 7 or 8, characterized in that the composition is co-administered with a second agent.
[0010]
10. Use according to claim 9, characterized in that the composition and the second agent are administered concurrently.

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法律状态:
2020-09-08| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-09-15| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-09-29| B25A| Requested transfer of rights approved|Owner name: ISIS PHARMACEUTICALS, INC. (US) |

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2020-10-20| B25D| Requested change of name of applicant approved|Owner name: IONIS PHARMACEUTICALS, INC. (US) |

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2021-03-23| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-03-30| B15K| Others concerning applications: alteration of classification|Free format text: AS CLASSIFICACOES ANTERIORES ERAM: C07H 21/04 , C12N 15/11 Ipc: C12N 15/113 (2010.01), C12N 15/11 (2006.01), C07H |

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2021-04-20| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-07-06| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-09-14| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 10/09/2010, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

优先权:

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申请号 | 申请日 | 专利标题

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US24185309P| true| 2009-09-11|2009-09-11|

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US61/241,853|2009-09-11|

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PCT/US2010/048532|WO2011032045A1|2009-09-11|2010-09-10|Modulation of huntingtin expression|

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